Chimeric polypeptides, polynucleotides encoding same, cells expressing same and methods of producing same

ABSTRACT

A plant produced chimeric polypeptide is provided. The plant produced chimeric polypeptide comprising:
     (i) a first domain which comprises a TNF Alpha binding domain of a TNF receptor, and   (ii) a second domain which comprises an Fc domain of an immunoglobulin, wherein the first domain and the second domain are N-terminally to C-terminally respectively sequentially translationally fused and wherein the chimeric polypeptide specifically binds TNF Alpha.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/849,963 filed on Sep. 10, 2015 which claims the benefit of priorityunder 35 USC § 119(e) of U.S. Provisional Patent Application No.62/048,932 filed on Sep. 11, 2014, the contents of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 74194SequenceListing.txt, created on Jun. 12,2018, comprising 87,059 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to chimericpolypeptides, polynucleotides encoding same, cells expressing same andmethods of producing same.

Tumor necrosis factor alpha (TNFα) is an important, pro-inflammatorycytokine mediating the regulation of diverse inflammatory, infectiousand immune-related processes and diseases, TNFα being considered themost important mediator responsible for inflammatory pathology.

TNF-alpha is a 17 kD molecular weight protein, initially synthesized asa transmembrane protein arranged in stable trimers, then cleaved bymetalloprotease-TNF alpha converting enzyme (TACE) to form thehomotrimeric soluble TNF (sTNF) which engages to its cognate receptors(TNFRI, p55 and TNFRII, p75), expressed ubiquitously. The ubiquitous TNFreceptors provide the basis for the wide variety of TNF-alpha mediatedcellular responses.

TNF-alpha induces a wide variety of cellular responses, many of whichresult in deleterious consequences, such as cachexia (loss of fat andwhole body protein depletion, leading to anorexia, common in cancer andAIDS patients) and septic shock. Elevated secretion of TNF-alpha hasbeen implicated in a variety of human diseases including diabetes,allograft rejection, sepsis, inflammatory bowel diseases, osteoporosis,in many autoimmune diseases such as multiple sclerosis, rheumatoidarthritis, psoriasis, psoriatic arthritis, hypersensitivity, immunecomplex diseases, and even in malaria, cancer and lung fibrosis.

The biological effect of TNFα is mediated by the two distinct receptors.TNF-alpha receptors, when shed from mononuclear cells, lower theTNF-alpha levels by “mopping up” and acting as natural inhibitorsNeutralization of TNFα by specific antibodies and decoy receptors hasbecome a common strategy for regulation of TNFα mediated toxicity.

To date, five protein-based TNFα antagonists have been approved by theUS FDA for clinical use: Cimzia (Certolizumab pegol), a TNFmAb Fab′fragment—PEG conjugate; Remicade (Infliximab), a TNF rmAB; Humira(Adalimumab), a TNF rmAB, Simponi™ (Golimumab), aTNF human monoclonalantibody and etanercept, a fusion protein of soluble 75 kDa TNFαreceptors fused to the Fc fragment of human IgG (registered as Enbrel™).

Etanercept is indicated for rheumatoid arthritis (RA) and otherarthritic indications such as juvenile idiopathic arthritis (JIA),psoriasis and Ankylosing Spondylitis (AS). Rheumatoid arthritis (RA) isa chronic disease that affects approximately five million people WorldWide. Nearly 500,000 patients worldwide across indications are treatedwith Enbrel. Enbrel sales in 2010 were 7.8 billion dollars and the totalanti-TNF market amounted to 24.04 Billion dollars. Clinical trials ofEnbrel therapy, current or completed, include such diverse indicationsas adult respiratory distress syndrome, pemphigus, Alzheimer's disease,Behcet's syndrome, HIV, myocardial infarct, knee joint synovitis, lupusnephritis, lichen planus, systemic amyloidosis, sciatica, vitiligo,chronic fatigue syndrome, anorexia, TMJ, asthma, bronchitis, diabetes,myelodysplastic disease and others.

Enbrel is currently produced in mammalian cells. The safety ofbiopharmaceuticals has recently come to the forefront for both patientsand health care providers due to outbreaks of emerging pathogens, mostnotably HIV, HCV, Cruezfeld-Jacob's Disease, West Nile Virus and SARS,in multiple regions of the world, emphasizing the risk of pathogentransmission through the use of human-or animal-derived raw materials,such as blood-derived products (serum, plasma cell medium components,etc) in the manufacture of biopharmaceuticals. For example,approximately half (!) of the hemophilia population contracted HIV untilidentification and screening for the virus became widespread.

Screening and testing have improved recently, reducing the threat ofpathogen transmission, but risks still remain from plasma-derivedadditives during recombinant manufacturing processes. In particular, therisk from unknown pathogens is significant, as these agents may appearin the blood supply in the future and could have a significant impact onsafety of mammalian-cell-based biopharmaceuticals. Of particular concernare biopharmaceutical drugs which require repeated, regularadministrations, specifically via injection, increasing the cumulativerisk to the patient.

However, eliminating animal-derived components from media cansignificantly alter culture performance as well as post-translationalprotein modifications. The glycosylation pattern of an antibody moleculecan affect its structural integrity, thus influencing its biologicalfunction, physicochemical properties and pharmacokinetics, altering bothefficacy and safety, particularly immunogenicity. Although no majoroutbreaks have occurred in recent years, it is still critical to reducedependence on blood and plasma components in the manufacture ofbiopharmaceuticals. Conversely, recombinant protein production inmammalian cell culture is unsafe due to xeno contaminations. In 2009Genzyme was forced to temporarily close its main factory because ofviral contamination. It did not restore full supplies of the drugs until2011. Due to the shortage in the only approved drug for Fabry patientsin the US, some people with Fabry disease have suffered heart or kidneyproblems and one or more may have even died because of the shortage.

Biopharmaceuticals, including modified human proteins, can be producedin transgenic plants in order to address problems of safety, viralinfections, immune reactions, production yield and cost. U.S. Pat. No.6,391,638 and PCT WO2008/135991 teach bioreactor devices forcommercial-scale production of recombinant polypeptides from plant cellculture. U.S. Pat. No. 7,951,557, U.S. patent application Ser. Nos.10/554,387 and 11/790,991 teach construction and expression of nucleicacid vectors for recombinant expression of human proteins in plantcells. PCT WO2007/010533 teaches the expression of recombinant humanpolypeptides in plant cells, for enteral administration.

Additional background art includes: U.S. Pat. No. 7,915,225 to Finck etal, U.S. patent application Ser. Nos. 13/021,545 and 10/853,479 to Fincket al, U.S. patent application Ser. No. 11/906,600 to Li et al, U.S.patent application Ser. No. 10/115,625 to Warren et al and U.S. patentapplication Ser. No. 11/784,538 to Gombotz et al.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a plant produced chimeric polypeptide comprising:

(i) a first domain which comprises a TNFα binding domain of a TNFreceptor, and(ii) a second domain which comprises an Fc domain of an immunoglobulin,wherein the first domain and the second domain are N-terminally toC-terminally respectively sequentially translationally fused and whereinthe chimeric polypeptide specifically binds TNFα.

According to an aspect of some embodiments of the present inventionthere is provided a chimeric polypeptide comprising:

(i) a first domain which comprises a TNFα binding domain of a TNFreceptor;(ii) a second domain which comprises an Fc domain of an immunoglobulin;and(iii) a third domain comprising an endoplasmic reticulum retentionsignal;wherein the first domain, second domain and third domain areN-terminally to C-terminally respectively sequentially translationallyfused and wherein the chimeric polypeptide specifically binds TNFα.

According to some embodiments of the invention, the polypeptidecomprises an additional domain encoding an endoplasmic reticulum signalpeptide translationally fused N-terminally to the first domain.

According to some embodiments of the invention, the signal peptide is aplant signal peptide.

According to some embodiments of the invention, the plant signal peptideis as set forth in SEQ ID NO: 4.

According to some embodiments of the invention, the first domain is200-250 amino acids long.

According to some embodiments of the invention, the first domaincomprises the amino acid sequence LCAP (SEQ ID NO: 11) and VFCT (SEQ IDNO: 12).

According to some embodiments of the invention, the first domain furthercomprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13) orLPAQVAFTPYAPEPGSTC (SEQ ID NO: 17).

According to some embodiments of the invention, the first domain is asset forth in SEQ ID NO: 2.

According to some embodiments of the invention, the immunoglobulin isIgG₁.

According to some embodiments of the invention, the second domain is asset forth in SEQ ID NO: 9.

According to some embodiments of the invention, the polypeptide is asset forth in SEQ ID NO: 6.

According to some embodiments of the invention, the polypeptide is asset forth in SEQ ID NO: 7, 204 or 205.

According to some embodiments of the invention, the polypeptide ispurified to at least 98% homogeneity.

According to some embodiments of the invention, the polypeptide iscapable of inhibiting TNFα-induced apoptosis.

According to some embodiments of the invention, the polypeptidecomprises a plant-specific glycan.

According to some embodiments of the invention, the plant-specificglycan is selected from the group consisting of a core xylose and a coreα-(1,3) fucose.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence encoding the polypeptide.

According to an aspect of some embodiments of the present inventionthere is provided a codon usage of the nucleic acid sequence isoptimized for Nicotinia tabaccum.

According to an aspect of some embodiments of the present inventionthere is provided the isolated polynucleotide as set forth in SEQ ID NO:5.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid expression construct comprising anucleic acid sequence encoding the polynucleotide and a cis-actingregulatory element active in a plant cell.

According to an aspect of some embodiments of the present inventionthere is provided the cis-acting regulatory element is a promoter.

According to an aspect of some embodiments of the present inventionthere is provided a plant cell comprising the nucleic acid construct.

According to an aspect of some embodiments of the present inventionthere is provided the plant cell is a Nicotiana tabacum plant cell.

According to an aspect of some embodiments of the present inventionthere is provided the Nicotiana tabacum L. cv plant cell is a BrightYellow (BY-2) cell.

According to an aspect of some embodiments of the present inventionthere is provided the plant cell is lyophilized.

According to an aspect of some embodiments of the present inventionthere is provided a plant cell suspension culture comprising the plantcell.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeingredient the polypeptide and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising as an activeingredient the plant cell and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a TNFα-associated medicalcondition in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of thepolypeptide, thereby treating the TNFα-associated medical condition inthe subject.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a TNFα-associated medicalcondition in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of theplant cells, thereby treating the TNFα-associated medical condition inthe subject.

According to an aspect of some embodiments of the present inventionthere is provided the polypeptide for use in treating a TNFα-associatedmedical condition in a subject.

According to some embodiments of the invention, the plant cells are foruse in treating a TNFα-associated medical condition in a subject.

According to an aspect of some embodiments of the present inventionthere is provided a use of the polypeptide in treating a TNFα-associatedmedical condition in a subject.

According to an aspect of some embodiments of the present inventionthere is provided a use of the plant cells in treating a TNFα-associatedmedical condition in a subject.

According to some embodiments of the invention, the medical condition isan inflammatory disease.

According to some embodiments of the invention, the medical condition isan autoimmune disease.

According to some embodiments of the invention, the medical condition isselected from the group consisting of rheumatoid arthritis, ankylosingspondyloarthritis, plaque psoriasis and juvenile idiopathic arthritis.

According to some embodiments of the invention, the medical condition isselected from the group consisting of rheumatoid arthritis, inflammatorybowel disease, short bowel syndrome, sepsis, endotoxic shock, AIDS,endometriosis, psoriasis, cardiovascular disease, cancer, vitiligo,arthritis, rheumatoid polyarthritis, psoriatic rheumatism, ankylosingspondyloarthritis, plaque psoriasis, juvenile idiopathic arthritis,polyarticular juvenile idiopathic arthritis, psoriasis arthritis,Wegener's disease (granulomatosis), Crohn's disease, short bowelsyndrome, ulcerative cholitis, chronic obstructive pulmonary disease(COPD), Hepatitis C, asthma, cachexia, atopic dermatitis. Alzheimer'sdisease, hepatic encephalopathy, ADHD, chronic fatigue syndromedermatitis herpetiformis (Duhring's disease), contact dermatitis,urticaria (including chronic idiopathic urticaria), autoimmuneblistering diseases, including pemphigus vulgaris, bullous pemphigoid,myesthenia gravis, sarcoidosis, including pulmonary sarcoidosis,scleroderma, reactive arthritis, hyper IgE syndrome, multiple sclerosisand idiopathic hypereosinophil syndrome, and allergy.

According to some embodiments of the invention, the medical condition isan inflammatory bowel disease.

According to some embodiments of the invention, the inflammatory boweldisease is ulcerative colitis or Crohn's disease.

According to some embodiments of the invention, the plant cells areformulated for oral administration.

According to some embodiments of the invention, the polypeptide isformulated for parenteral administration.

According to some embodiments of the invention, the plant cells areformulated for enteral administration and wherein the medical conditionis not an obesity, metabolic syndrome, diabetes and a liver disease ordisorder.

According to an aspect of some embodiments of the present inventionthere is provided a method of producing the polypeptide, comprising:

providing a cell as described herein; and

culturing the cell so as to produce the polypeptide.

According to some embodiments of the invention, the method furthercomprises isolating the polypeptide from the cell.

According to some embodiments of the invention, the cell is an isolatedcell cultured in a plant cell culture medium.

According to some embodiments of the invention, the culturing isperformed in a disposable bioreactor.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a TNFα-associated medicalcondition in a subject in need thereof, the method comprising orallyadministering to the subject a therapeutically effective amount of theplant cells of claim 23, wherein the therapeutically effective amount ofthe polypeptide in the plant cells comprises 0.02-0.27 mg/kg, therebytreating the TNFα-associated medical condition in the subject.

According to an aspect of some embodiments of the present inventionthere is provided use of the plant cells of claim 23 in the manufactureof a medicament identified for the treatment of a TNFα-associatedmedical condition, wherein a therapeutically effective amount of thepolypeptide in the plant cells is 0.02-0.27 mg/kg, wherein the plantcells are formulated for oral administration.

According to some embodiments of the invention, the therapeuticallyeffective amount comprises 0.02-0.12 mg polypeptide/kg.

According to some embodiments of the invention, the therapeuticallyeffective amount comprises 0.12-0.27 mg polypeptide/kg.

According to an aspect of some embodiments of the present inventionthere is provided an oral dosage form comprising the plant cells ofclaim 23 comprising 1-20 mg of the polypeptide.

According to some embodiments of the invention, the oral dosage formcomprises 1-10 mg of the polypeptide.

According to some embodiments of the invention, the oral dosage formcomprises 10-20 mg of the polypeptide.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a schematic illustration of the amino acid sequence of plantrecombinant human (prh) TNFR2:Fc (also termed herein PRX-106, SEQ IDNO:6). prh TNFR2:Fc cDNA for expression in BY2 cells was assembled witha signal peptide for targeting the fusion polypeptide composed of theTNF-binding moiety of the TNF receptor and FC protein to the secretorypathway. Colour code for the amino acids sequence: the signal peptide iscoloured in yellow; the TNF receptor portion is coloured in black(green); the Fc portion of IgG1 is in blue; ER retention signal in red.

FIGS. 2A-2B show comparison of PRH TNFR2:FC and Commercial Enbrel byWestern-blot. prh TNFR2:Fc (lane 1) and commercial Enbrel (lane 2) wereanalyzed under reducing conditions (panel A) and non-reducing conditions(panel B) by 12% and 8% Tris-Glycine SDS-PAGE, respectively. Membraneswere blotted with an anti FC antibody (upper panel) and with an antiTNFR2 antibody (lower panel). Molecular weight marker is shown in rightlanes. Lane 1: prh TNFR2:FC; Lane 2:commercial Enbrel.

FIG. 3 is a graph showing TNFα binding by prh TNFR2:Fc and commercialEnbrel by quantitative non radioactive assay for prh TNFR2:Fc bindingactivity and molecular integrity. An ELISA plate pre-coated withantibodies against TNFα, was incubated with TNFα followed by exposure tocommercial Enbrel and supernatant from BY2 cells expressing prhTNFR2:Fc. Serial dilutions of both tested items are shown in the X axis.Fc portion of the molecule was detected with Goat anti human IgG Fc HRP.

FIG. 4 is an image showing screening of individual cell lines forexpression of prh TNFR2:Fc by Western blot analysis.

FIGS. 5A-5F are images showing TNFα cytotoxicity in A375 cells in thepresence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.FIG. 5A—untreated Cultured A375 cells; FIG. 5B—treated with TNFα; FIG.5C—TNFα exposed cells treated with prh TNFR2:Fc (3.125 ng/ml); FIG.5D—TNFα exposed cells treated with commercial Enbrel (3.125 ng/ml); FIG.5E—TNFα exposed cells treated with prh TNFR2:Fc (100 ng/ml); FIG.5F—TNFα exposed cells treated with commercial Enbrel (100 ng/ml).

FIG. 5G is a bar graph showing TNFα cytotoxicity in A375 cells in thepresence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.

FIGS. 6A-6F are images showing TNFα cytotoxicity in L929 cells in thepresence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.FIG. 6A—untreated Cultured L929 cells; FIG. 6B—treated with TNFα; FIG.6C—TNFα exposed cells treated with prh TNFR2:Fc (3.125 ng/ml); FIG.6D—TNFα exposed cells treated with commercial Enbrel (3.125 ng/ml); FIG.6E—TNFα exposed cells treated with prh TNFR2:Fc (100 ng/ml); FIG.6F—TNFα exposed cells treated with commercial Enbrel (100 ng/ml).

FIG. 6G is a bar graph showing TNFα cytotoxicity in L929 cells in thepresence of prh TNFR2:Fc or commercial Enbrel by MTT viability assay.

FIGS. 7A-7B are graphs showing body weight changes following TNBSchallenge. Mice were orally administered with prh TNFR2:Fc 6 hours afterTNBS induction. For four consecutive days body weights were determineddaily (means±SE). FIG. 7A—Average weight loss at day four followingtreatment with TNBS, presented in % loss from original weight. column1-saline control (n=15); column 2—Mock-host plant control cells (BY2−;n=15); column 3-PRX-106-plant cells expressing recombinant TNFR2:Fc(doseI) (n=15); column 4-PRX-106-plant cells expressing recombinantTNFR2:Fc (dose II) (n=7); column 5-Dexametason treated mice (n=10);column 6-control mice (n=5). FIG. 7B—Average weight loss during fourdays following treatment with TNBS, presented in % loss from originalweight. Note oral treatment with plant cells expressing recombinantTNFR2:Fc attenuated body weight reduction.

FIG. 8 is a bar graph showing that oral administration of plant cellsexpressing TNFR2:Fc inhibits TNBS-induced colonic shorting. The micewith colitis were orally administered with plant cells expressingTNFR2:Fc for four consecutive days after TNBS induction and the colonlengths were determined at day 4 (means±SE). From left to right: Column1—control mice (n=5); column 2—saline control (n=15); column 3—Mock-hostplant control cells (BY2−; n=15); column 4—PRX-106-plant cellsexpressing recombinant TNFR2:Fc (doseI) (n=15); column 5—PRX-106-plantcells expressing recombinant TNFR2:Fc (dose II) (n=7); column6—Dexametason treated mice (n=10).

FIG. 9 is a bar graph showing that oral administration of plant cellsexpressing TNFR2:Fc improved the macroscopic futures of TNBS-inducedcolitis. Total macroscopic inflammation scores (Wallace score) incontrol and treated rats at the endpoint of the experiment (means±SE).Column 1—control mice (n=5); column 2—saline control (n=15); column3—Mock-host plant control cells (BY2−; n=15); column 4—PRX-106-plantcells expressing recombinant TNFR2:Fc (doseI) (n=15); column5—PRX-106-plant cells expressing recombinant TNFR2:Fc (dose II) (n=7);column 6—Dexametason treated mice (n=10).

FIGS. 10A-10C are bar graphs showing serum cytokine content in micetreated by oral administration of plant cells expressing TNFR2:Fc asmeasured by a cytokine antibody array. Sera from groups treated withMock-host plant control cells (BY2−) (n=15) and Plant cells expressingrecombinant TNFR2:Fc protein dose I (n=15), and dose II (n=7) werecollected and subjected to cytokine magnetic Luminex assay. TNBS-salinecontrol (n=15); Mock-host plant control cells (BY2−; n=15);PRX-106-plant cells expressing recombinant TNFR2:Fc (doseI) (n=15);PRX-106-plant cells expressing recombinant TNFR2:Fc (dose II) (n=7);Dexametason treated mice (n=10); control mice (n=5).

FIGS. 11A-11B show serum cytokine content by cytokine Antibody array.

FIG. 11A—Sera from groups treated with Mock-host plant control cells(BY2−) (n=15) and plant cells expressing recombinant TNFR2:Fc proteindose I (n=15), and dose II (n=7) were pooled, collected and subjected tocytokine antibody array analysis.

FIG. 11B—Cytokine quantification of array. Results indicate thattreatment with PRX-106 reduced level of inflammatory mediators likegranulocyte colony-stimulating factor G-CSF, macrophagecolony-stimulating factor (M-CSF), potentially indicating reducedsystemic inflammation by lowering systemic recruitment of bone marrowderived cells from the bloodstream.

FIG. 12 is a bar graph showing expansion of splenic Treg population inanimals treated with plant cells expressing recombinant TNFR2:Fc. Spleenof Balb/c mice, treated with PRX-106 during TNBS induced colitis wereanalyzed for the percentages of CD4+CD25+Foxp3+, bars indicate SE.Column 1—saline control (n=15); column 2—Mock-host plant control cells(BY2−; n=15); column 3—PRX-106-plant cells expressing recombinantTNFR2:Fc (doseI) (n=15); column 4—PRX-106-plant cells expressingrecombinant TNFR2:Fc (dose II) (n=7); column 5—Dexametason treated mice(n=10); column 6—control mice (n=5).

FIGS. 13A-13B are graphs showing body weight changes following DSSchallenge. The mice with colitis were orally administered with plantcells expressing TNFR2:Fc for seven consecutive days 24 hours after DSSinduction and the body weights of mice were determined (means±SE). FIG.13A—Average weight loss at day ten following treatment with DSS,presented in % loss from original weight. column 1—saline control(n=10); column 2—Mock-host plant control cells (BY2−; n=10); column3—oral administration of plant cells expressing recombinant TNFR2:Fc(n=10); column 4—control mice (n=5). FIG. 13B—Average weight loss duringten days following treatment with DSS, presented in % loss from originalweight. Note oral treatment with PRX-106-plant cells expressingrecombinant TNFR2:Fc attenuated body weight reduction. * P<0.05, **P<0.01, *** P<0.001.

FIGS. 14A-14B show that oral administration of TNFR2:Fc inhibitedDSS-induced colonic shorting. The mice with colitis were orallyadministered with plant cells expressing recombinant TNFR2:Fc for 7consecutive days after DSS induction and the colon lengths weredetermined at day 10 (means±SE). FIG. 14A—Column 1—saline control(n=10); column 2—Mock-host plant control cells (BY2−; n=10); column3—plant cells expressing recombinant TNFR2:Fc (n=10); column 4—controlmice (n=5). FIG. 14B—Representative photograph of colons, ten days afterthe induction of DSS colitis.

FIGS. 15A-15C are graphic presentations of cytokine profile in colonsobtained from treated mice. Cytokine secretion by ex vivo—cultured punchbiopsies harvested from the colon of Column 1—DSS treated mice receivingsaline control (n=10); column 2—DSS treated mice receiving Mock-hostplant control cells (BY2−; n=10); column 3—DSS treated mice receivingplant cells expressing 30 μg recombinant TNFR2:Fc (n=10); column4—control untreated mice (n=5).

FIGS. 16A-16C are graphic presentations of serum cytokine contentassayed by cytokine Antibody array. Sera from mice treated withMock-host plant control cells (BY2−) n=15) and Plant cells expressingrecombinant TNFR2:Fc protein (n=10), were collected and subjected tocytokine magnetic Luminexas say. Column 1—saline control (n=10); column2—Mock-host plant control cells (BY2−; n=10); column 3—oraladministration of plant cells expressing recombinant TNFR2:Fc (n=10);column 4—control mice (n=5). * P<0.05.

FIGS. 17A-17B show that therapeutic treatment with orally administeredplant cells expressing recombinant TNFR2:Fc reduces the severity ofDSS-induced colitis. FIG. 17A—Representative histological sections wereexamined microscopically after H&E staining with magnification ×40 and×100. The images are representative of at least seven mice per group.FIG. 17B—The effect of orally administered plant cells expressingrecombinant TNFR2:FC on histological colitis score was determined. Whitesquare—plant cells expressing recombinant TNFR2:Fc (n=7), Graysquare—Mock-host plant control cells (BY2−; n=10), Black square—salinecontrol (n=8). ** P<0.01, *** P<0.001.

FIG. 18 is a bar graph showing pharmacokinetics of TNFR2:Fc in rat sera.Oral administration of plant cells expressing recombinant TNFR2:Fc wasinitiated by free feeding. Rats (n=6) received plant cells expressingrecombinant TNFR2:Fc. Negative controls received the same volumes ofhost BY2(−) plant.

FIG. 19 is a bar graph showing pharmacokinetics of TNFR2:Fc in rat serafollowing oral administration of plant cells expressing recombinantTNFR2:Fc by gavage. Rats (n=6) received plant cells expressingrecombinant TNFR2:Fc. Negative controls received the same volumes ofhost BY2(−) plant.

FIG. 20 shows the PRX-106 sequence (SEQ ID NO: 6) elucidated bymass-spec (green shown 84.8% coverage).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to chimericpolypeptides, polynucleotides encoding same, cells expressing same andmethods of producing same.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Etanercept is a tumor necrosis factor (TNF) blocker indicated for anumber of inflammatory conditions such as rheumatoid arthritis,polyarticular juvenile idiopathic arthritis, plaque psoriasis, psoriaticarthritis and ankylosing spondylitis. Etanercept is produced byrecombinant DNA technology in Chinese hamster ovary mammalian cellexpression system. The production of recombinant proteins in mammaliancell systems is hampered by cellular fragility and the complexnutritional requirements of cells and the possible contamination of thefinal product with virus or prions.

Whilst reducing the present invention to practice, the present inventorshave constructed an expression vector for recombinant expression ofEnteracept (hereinafter, prh TNFR2:Fc) in plant cells, transformedtobacco cells with the vector, and have isolated catalytically activeprotein from the cell cultures. The expressed recombinant proteinretains its TNFα binding activity and has shown favorable catalyticactivity as evidenced by its apoptosis regulatory activity.

In-vivo studies in animal models for inflammation e.g., inflammatorybowel disease, support the efficacy of the protein and specifically anoral formulation thereof in treatment of said indications.

Specifically, the effect of oral administration of prh TNFR2:Fc in plantcells on colitis was examined in two chemically-induced mouse models forIBD: (i) induced by intra-rectal administration of the covalentlyreactive reagents TNBS/oxazolone; and (ii) induced by injections ofdextran sodium sulfate. The results shown in Examples 3A-B, below,illustrate that oral administration of plant cells expressing prhTNFR2:Fc ameliorated weight loss, significantly increased colon length,reduced colon damage (as determined histopathologically), reduced thelevel of secreted pro-inflammatory cytokines in-situ and in sera, andelicited splenic Treg expansion. Oral administration of plant cellsexpressing the chimeric polypeptide shifted the T cells populationprofile following treatment. Accordingly, there is a shift in therelative number of regulatory T cell and NKT cell populations uponadministration of plants cells expressing the chimeric polypeptide asshown using various FACS analysis markers e.g, CD4⁺CD25⁺CD8⁺CD25⁺.CD4⁺CD25⁺Foxp3⁺ and CD3⁺CD56⁺ in the treated subjects.

These results conclusively show that prh TNFR2 is biologically active asan anti-inflammatory agent. The present results further support a rolefor orally administered plant cells expressing recombinant TNFR2:Fc asan anti-inflammatory agent with the capacity to ameliorate IBD.

The present inventors have further performed a toxicology study inanimals. The results presented in Example 4 below, show that oraladministration of plant cells expressing prh TNFR2:Fc is safe and welltolerated.

Thus, according to an aspect of the present invention, there is provideda plant produced chimeric polypeptide comprising:

(i) a first domain which comprises a TNFα binding domain of a TNFreceptor; and(ii) a second domain which comprises an Fc domain of an immunoglobulin,wherein the first domain and the second domain are N-terminally toC-terminally respectively sequentially translationally fused and whereinthe chimeric polypeptide specifically binds TNFα.

As used herein the term “plant produced” refers to the chemicalsignature associated with plant expression, including, but not limitedto, host cell impurities in the preparation which comprises the chimericpolypeptide and glycosylation patterns on the chimeric polypeptide perse.

As used herein the term “chimeric polypeptide” refers to a proteincreated through the joining of two or more individual coding sequenceswhich originally code for separate proteins. Translation of thesynthetic (non-naturally occurring) nucleic acid sequence results in asingle chimeric polypeptide with functional properties derived from eachof the original proteins. Such recombinant fusion proteins are createdartificially by recombinant DNA technology.

As used the term “TNFα” refers to Tumor necrosis factor-alpha (TNF,cachexin, or cachectin) that is a cytokine involved in systemicinflammation and a member of a group of cytokines that stimulate theacute phase reaction. TNFα is produced primarily by activatedmacrophages (M1), although it can be produced by many other cell typesas CD4+ lymphocytes, NK cells and neurons. The protein is encoded byTNFA gene and has the Ref_seq number: NP_000585. The protein is known tostimulate an inflammatory response (pro-inflammatory cytokine).

As used herein the term “TNF receptor” or “TNFR” refers to a polypeptidewhich is capable of binding TNFα in a specific manner e.g., Kd below10⁻⁵ M. According to a specific embodiment, the TNFR is membrane bound.

The first domain is thus composed of at least the TNF binding domain ofa TNF receptor (TNFR). The first domain is a soluble protein. Thusaccording to a specific embodiment, the first domain and even the entirechimeric polypeptide are soluble proteins which are not membraneanchored.

Soluble forms of TNFRs may include monomers, fusion proteins (alsocalled “chimeric proteins), dimers, trimers or higher order multimers.In certain embodiments of the invention, the soluble TNFR derivative isone that mimics the 75 kDa TNFR or the 55 kDa TNFR and that binds toTNFα. in vivo. The soluble TNFR mimics of the present invention may bederived from TNFRs p55 or p75 or fragments thereof. TNFRs other than p55and p75 also are useful for deriving soluble TNFR for treating thevarious medical disorders described herein, such for example the TNFRthat is described in WO 99/04001. Soluble TNFR molecules used toconstruct TNFR mimics include, for example, analogs or fragments ofnative TNFRs having at least 20 amino acids, that lack the transmembraneregion of the native TNFR, and that are capable of binding TNFα. Suchsoluble forms of TNFR compete for TNFα with the receptors on the cellsurface, thus inhibiting TNFα from binding to cells, thereby preventingit from manifesting its biological activities. Binding of soluble TNFRsto TNFα can be assayed using ELISA or any other convenient assay.

According to a specific embodiment, the first domain is derived fromTNFR2. (e.g., AAA36755).

According to an embodiment of the invention, the first domain is 200-250amino acids long.

According to a specific embodiment, the first domain comprises the aminoacid sequence LCAP (SEQ ID NO: 11) and VFCT (SEQ ID NO: 12).

According to a specific embodiment, the first domain comprises the aminoacid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13), or LPAQVAFTPYAPEPGSTC(SEQ ID NO: 17).

According to a specific embodiment, the first domain is as set forth inSEQ ID NO: 2 (encoded by SEQ ID NO: 1).

As used herein “an Fc domain of an immunoglobulin” refers to a region ofa heavy chain of an antibody, typically comprising at least 2 constantdomains (e.g., CH2 and CH3 domains, as these terms are defined in theart) of the heavy chain. The Fc domain may be obtained, for example, inthe form of a dimer, by digestion of an antibody by papain. A dimer ofFc domain polypeptides, connected by disulfide bonds, forms the “tail”region of an antibody. As is known in the art, Fc domains of someclasses of antibodies may be in the form of multimers. Thus, the Fcdomain is optionally monomeric, optionally dimeric and optionallymultimeric. Optionally, the polypeptide described herein is in the formof a dimer, the polypeptide comprising an Fc dimer, or in the form of amultimer, the polypeptide comprising an Fc multimer.

The Fc domain may encompass modified forms of a native Fc domain (i.e.,a domain which occurs naturally in an antibody), for example,polypeptides having at least 90% homology, optionally at least 95%homology, and optionally at least 98% homology, to a native Fc domain.Modified Fc domains are described, for example, in International PatentApplications WO 97/34631 and WO 96/32478.

Optionally, a native Fc is modified so as to remove sites which providestructural features or biological activity that are not required forembodiments of the present invention. Examples of such sites includeresidues that affect or are involved in disulfide bond formation,incompatibility with a selected host cell, N-terminal heterogeneity uponexpression in a selected host cell, glycosylation, interaction withcomplement, binding to an Fc receptor (other than a neonatal Fcreceptor), and/or antibody-dependent cellular cytotoxicity.

The polypeptide according to embodiments of the present invention mayalso comprise a fragment of an Fc domain. Optionally, the fragmentcomprises at least 20%, optionally at least 50%, and optionally at least80% of an Fc domain, as defined hereinabove.

The Fc domain or fragment thereof optionally includes a binding site fora neonatal Fc receptor (FcRn). This is of particular significance whenadministering the chimeric polypeptide via an enteral route.

According to one embodiment, attachment of an Fc domain or a fragmentthereof to the first domain results in a polypeptide having a longerhalf-life in vivo than the first domain per se. This may be due to thelong serum half-life of the Fc domain (which may be due to salvage ofthe Fc via binding to FcRn) and/or due to the greater size of thepolypeptide in comparison to the first domain per se, which reducesclearance from the bloodstream by glomerular filtration. According toanother embodiment, the resulting polypeptides have reducedimmunogenicity as compared to the first domain per se.

According to optional embodiments, the Fc domain or fragment thereof isa human Fc domain (e.g., derived from a human antibody) or fragmentthereof.

According to exemplary embodiments, the Fc domain (or fragment thereof)is an IgG (e.g., IgG1) Fc domain (or fragment thereof).

According to a specific embodiment, the second domain is as set forth inSEQ ID NO: 9 (encoded by SEQ ID NO: 8).

Thus, the second domain of the chimeric polypeptide comprises at least aportion of a constant immunoglobulin domain, e.g. a constant heavyimmunoglobulin domain or a constant light immunoglobulin domain.Preferably, the second domain comprises at least a portion of a constantheavy immunoglobulin domain. The constant heavy immunoglobulin domain ispreferably an Fc fragment comprising the CH2 and CH3 domain and,optionally, at least a part of the hinge region. The immunoglobulindomain may be an IgG, IgM, IgD or IgE immunoglobulin domain or amodified immunoglobulin domain derived, therefrom. Preferably, thesecond domain comprises at least a portion of a constant IgGimmunoglobulin domain. The IgG immunoglobulin domain may be selectedfrom IgG1, IgG2, IgG3 of IgG4 domains or from modified domains such asare described in U.S. Pat. No. 5,925,734. The immunoglobulin domain mayexhibit effector functions. In some embodiments, however, modifiedimmunoglobulin domains having modified, e.g. at least partially deleted,effector functions may be used. Thus for example, the receptor.

According to an embodiment of the invention the chimeric fusion of thefirst domain and the second domain forms Etanercept (Immunex) having SEQID NO: 10.

It will be appreciated that the species origin of the first domain andthe second domain is selected according to the treated subject. Thus,according to a specific embodiment, the first domain and the seconddomain are of human origin or modified such that they don't incurimmunogenic reaction when administered to human subjects.

As used herein “Etanercept” and “Enbrel™” are interchangeably used todesignate the commercially available TNFR2:Fc by Immunex Corporation.Etanercept is a dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgG1. The Fc component of etanercept contains the constant heavy 2 (CH2)domain, the constant heavy 3 (CH3) domain and hinge region, but not theconstant heavy 1 (CH1) domain of human IgG1.

According to another embodiment of the invention there is provided achimeric polypeptide comprising:

(i) a first domain which comprises a TNFα binding domain of a TNFreceptor;(ii) a second domain which comprises an Fc domain of an immunoglobulin;and(iii) a third domain comprising an endoplasmic reticulum retentionsignal;wherein the first domain, second domain and third domain areN-terminally to C-terminally respectively sequentially translationallyfused and wherein the chimeric polypeptide specifically binds TNFα.

Thus, according to this aspect of the invention, the chimeric protein isexpressed such that it is retained in the endoplasmic reticulum (ER).According to a specific embodiment, at least a portion (e.g., 20% ormore) of the TNFR2:Fc molecules in the cell are retained in the ER.

As used herein, the term “endoplasmic reticulum retention signalpeptide” refers to a peptide sequence which, when present at the N- orC-terminus of a polypeptide, causes the polypeptide to be retrieved fromthe Golgi apparatus, and retained in the endoplasmic reticulum (seeRayon et al. Journal of Experimental Botany, Vol. 49, No. 326, pp.1463-1472, 1998; and Neumann, et al Annals of Botany, 2003; 92:167-180).In one embodiment, the endoplasmic reticulum retention signal peptide isHDEL (SEQ ID NO: 14), KDEL (SEQ ID NO: 15) or SEKDEL (SEQ ID NO: 16).

As mentioned, the first domain and second domain (and third domain whenpresent) are N-terminally to C-terminally respectively sequentiallytranslationally fused. This means that the first domain is locatedN-terminally to the second domain (the carboxy terminus of the firstdomain is translationally fused to the N-terminus of the second domain),and the second domain is located N-terminally of the third domain (thecarboxy terminus of the second domain is translationally fused to theN-terminus of the third domain). Thus, the second domain is practicallysandwiched by the first domain at the N-terminus and the third domain atthe C-teminus. Schematic presentation is as follows: first domain>seconddomain(>third domain) are orderly oriented from the N-terminus to theC-terminus (see FIG. 1). The linkage between the domains may be director indirect by the use of linkers such as peptide linkers.

The molecule may further comprise an additional domain which encodes foran endoplasmic reticulum signal sequence which is oriented upstream(N-terminally) of the first domain and translationally fused thereto.

As used herein “an endoplasmic reticulum (ER) signal peptide” refers toa signal sequence, leader sequence or leader peptide that is a short(e.g., 5-30 amino acids long) peptide present at the N-terminus of themajority of newly synthesized proteins that are destined towards thesecretory pathway.

According to a specific embodiment, the ER signal peptide is derived(taken) from a plant protein.

According to a specific embodiment, the endoplasmic reticulum signalpeptide is from N. plumbaginifolia Calreticulin protein.

According to a further specific embodiment, the signal peptide from N.plumbaginifolia Calreticulin protein is as set forth in SEQ ID NO: 4 andencoded by the nucleic acid sequence of SEQ ID NO: 3.

As used herein the term “translationally fused at the N-terminal” or“translationally fused at the C-terminal” refers to covalent attachmentof the indicated peptide via a peptide bond to the N-terminal orC-terminal amino acid of the respective domain typically as a result ofrecombinant expression.

According to a specific embodiment, the chimeric polypeptide is as setforth in SEQ ID NO: 6.

According to a specific embodiment, the chimeric polypeptide is as setforth in SEQ ID NO: 7, 204 or 205.

As mentioned the recombinant chimeric proteins of the invention areproduced in plant cells.

In order to express the polypeptide, an isolated polynucleotidecomprising a nucleic acid sequence encoding the chimeric polypeptide asdescribed herein is ligated into a “plant nucleic acid expressionconstruct”.

As used herein the term “plant nucleic acid expression construct” refersto a nucleic acid construct which includes the nucleic acid of someembodiments of the invention and at least one promoter for directingtranscription of nucleic acid in a host plant cell. Further details ofsuitable transformation approaches are provided hereinbelow.

According to some embodiments of the invention, there is provided anucleic acid expression construct comprising the nucleic acid sequenceof the invention, and a promoter for directing transcription of thenucleic acid sequence in a plant host cell.

As used herein the term “nucleic acid sequence” refers to a single ordouble stranded nucleic acid sequence which is isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

As used herein the phrase “complementary polynucleotide sequence” refersto a sequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.Such a sequence can be subsequently amplified in vivo or in vitro usinga DNA dependent DNA polymerase.

As used herein the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

As used herein the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis acting expression regulatoryelements.

According to some embodiments of the present invention, the nucleic acidsequences encoding the polypeptides of the present invention areoptimized for expression in plants. Examples of such sequencemodifications include, but are not limited to, an altered G/C content tomore closely approach that typically found in the plant species ofinterest, and the removal of codons atypically found in the plantspecies commonly referred to as codon optimization. In one embodiment,the codon usage of the nucleic acid sequence encoding the chimericpolypeptide is optimized for Nicotiana tabacuum or Nicotianabenthamiana.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1 N [(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants has been compiled usingthe data of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (Hypertext Transfer Protocol://World WideWeb (dot) kazusa (dot) or (dot) jp/codon/). The Codon Usage Databasecontains codon usage tables for a number of different species, with eachcodon usage table having been statistically determined based on the datapresent in Genbank.

By using such codon optimization tables to determine the most preferredor most favored codons for each amino acid in a particular species (forexample, rice), a naturally-occurring nucleotide sequence encoding aprotein of interest can be codon optimized for that particular plantspecies. This is effected by replacing codons that may have a lowstatistical incidence in the particular species genome withcorresponding codons, in regard to an amino acid, that are statisticallymore favored. However, one or more less-favored codons may be selectedto delete existing restriction sites, to create new ones at potentiallyuseful junctions (5′ and 3′ ends to add signal peptide or terminationcassettes, internal sites that might be used to cut and splice segmentstogether to produce a correct full-length sequence), or to eliminatenucleotide sequences that may negatively affect mRNA stability orexpression.

The desired encoding nucleotide sequence may already, in advance of anymodification, contain a number of codons that correspond to astatistically-favored codon in a particular plant species. Therefore,codon optimization of the native nucleotide sequence may comprisedetermining which codons, within the desired nucleotide sequence, arenot statistically-favored with regards to a particular plant, andmodifying these codons in accordance with a codon usage table of theparticular plant to produce a codon optimized derivative. A modifiednucleotide sequence may be fully or partially optimized for plant codonusage provided that the protein encoded by the modified nucleotidesequence is produced at a level higher than the protein encoded by thecorresponding naturally occurring or native gene. Construction ofsynthetic genes by altering the codon usage is described in for examplePCT Patent Application 93/07278.

Thus according to a specific embodiment, there is provided a Nicotiniatobaccum optimized sequence as set forth in SEQ ID NO: 5.

According to some embodiments of the invention, the nucleic acidsequence coding for the cimeric polypeptide is operably linked to acis-acting regulatory sequence active in plant cells, such as a plantpromoter sequence.

A coding nucleic acid sequence is “operably linked” to a regulatorysequence (e.g., promoter) if the regulatory sequence is capable ofexerting a regulatory effect on (e.g. effect on the expression of) thecoding sequence linked thereto.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention. Preferably the promoter is a constitutivepromoter, a tissue-specific, or an inducible promoter.

As used herein the phrase “plant-expressible” refers to a promotersequence, including any additional regulatory elements added thereto orcontained therein, is at least capable of inducing, conferring,activating or enhancing expression in a plant cell, tissue or organ,preferably a monocotyledonous or dicotyledonous plant cell, tissue, ororgan. Such a promoter can be constitutive, i.e., capable of directinghigh level of gene expression in a plurality of tissues, tissuespecific, i.e., capable of directing gene expression in a particulartissue or tissues, inducible, i.e., capable of directing gene expressionunder a stimulus, or chimeric, i.e., formed of portions of at least twodifferent promoters.

Examples of preferred promoters useful for the methods of someembodiments of the invention are presented in Table I, II, III and IV.

TABLE I Exemplary constitutive promoters for use in the performance ofsome embodiments of the invention Gene Source Expression PatternReference Actin constitutive McElroy et al, Plant Cell, 2: 163-171, 1990CAMV 35S constitutive Odell et al, Nature, 313: 810-812, 1985 CaMV 19Sconstitutive Nilsson et al., Physiol. Plant 100: 456-462, 1997 GOS2constitutive de Pater et al, Plant J Nov; 2(6): 837-44, 1992 ubiquitinconstitutive Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Ricecyclophilin constitutive Bucholz et al, Plant Mol Biol. 25(5): 837-43,1994 Maize H3 histone constitutive Lepetit et al, Mol. Gen. Genet. 231:276-285, 1992 Actin 2 constitutive An et al, Plant J. 10(1); 107-121,1996

TABLE II Exemplary seed-preferred promoters for use in the performanceof some embodiments of the invention Expression Gene Source PatternReference Seed specific genes seed Simon, et al., Plant Mol. Biol. 5.191, 1985; Scofield, etal., J. Biol. Chem. 262: 12202, 1987.;Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut albuminseed Pearson' et al., Plant Mol. Biol. 18: 235-245, 1992. legumin seedEllis, et al. Plant Mol. Biol. 10: 203-214, 1988 Glutelin (rice) seedTakaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa, et al.,FEBS Letts. 221: 43-47, 1987 Zein seed Matzke et al Plant Mol Biol,143).323-32 1990 napA seed Stalberg, et al, Planta 199: 515-519, 1996wheat LMW and endosperm Mol Gen Genet 216: 81-90, HMW, glutenin-1 1989;NAR 17: 461-2, Wheat SPA seed Albanietal, Plant Cell, 9: 171-184, 1997wheat a, b and g endosperm EMBO3: 1409-15, 1984 gliadins Barley ltrlpromoter endosperm barley B1, C, D endosperm Theor Appl Gen 98: 1253-62,hordein 1999; Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996Barley DOF endosperm Mena et al, The Plant Journal, 116(1): 53-62, 1998Biz2 endosperm EP99106056.7 Synthetic promoter endospermVicente-Carbajosa et al., Plant J. 13: 629-640, 1998 rice prolaminendosperm Wu et al, Plant Cell Physiology NRP33 39(8) 885-889, 1998rice-globulin Glb-1 endosperm Wu et al, Plant Cell Physiology 398)885-889, 1998 rice OSH1 embryo Sato et al, Proc. Nati. Acad. Sci. USA,93: 8117-8122 rice alpha-globulin endosperm Nakase et al. Plant Mol.Biol. REB/OHP-1 33: 513-S22, 1997 rice ADP-glucose endosperm Trans Res6: 157-68, 1997 PP maize ESR gene endosperm Plant J 12: 235-46, 1997family sorghum gamma- endosperm PMB 32: 1029-35, 1996 kafirin KNOXembryo Postma-Haarsma ef al, Plant Mol. Biol. 39: 257-71, 1999 riceoleosin Embryo Wu et at, J. Biochem., 123: 386, and aleuton 1998sunflower oleosin Seed (embryo Cummins, etal., Plant Mol. and dry seed)Biol. 19: 873-876, 1992

TABLE III Exemplary flower-specific promoters for use in the performanceof the invention Expression Gene Source Pattern Reference AtPRP4 flowerswww(dot)salus(dot)medium(dot)edu/mmg/ tierney/html chalene flowers Vander Meer, et al., Plant Mol. Biol. synthase 15, 95-109, 1990. (chsA)LAT52 anther Twell et al Mol. Gen Genet. 217: 240-245 (1989) apetala-3flowers

TABLE IV Alternative rice promoters for use in the performance of theinvention PRO # gene expression PR00001 Metallothionein Mte transferlayer of embryo + calli PR00005 putative beta-amylase transfer layer ofembryo PR00009 Putative cellulose synthase Weak in roots PR00012 lipase(putative) PR00014 Transferase (putative) PR00016 peptidyl prolylcis-trans isomerase (putative) PR00019 unknown PR00020 prp protein(putative) PR00029 noduline (putative) PR00058 Proteinase inhibitorRgpi9 seed PR00061 beta expansine EXPB9 Weak in young flowers PR00063Structural protein young tissues + calli + embryo PR00069 xylosidase(putative) PR00075 Prolamine 10 Kda strong in endosperm PR00076 allergenRA2 strong in endosperm PR00077 prolamine RP7 strong in endospermPR00078 CBP80 PR00079 starch branching enzyme 1 PR00080Metallothioneine-like ML2 transfer layer of embryo + calli PR00081putative caffeoyl-CoA 3-0 shoot methyltransferase PR00087 prolamine RM9strong in endosperm PR00090 prolamine RP6 strong in endosperm PR00091prolamine RP5 strong in endosperm PR00092 allergen RA5 PR00095 Putativemethionine embryo aminopeptidase PR00098 ras-related GTP binding proteinPR00104 beta expansine EXPB1 PR00105 Glycine rich protein PR00108metallothionein like protein (putative) PR00110 RCc3 strong root PR00111uclacyanin 3-like protein weak discrimination center/ shoot meristemPR00116 26S proteasome regulatory very weak meristem particle non-ATPasesubunit 11 specific PR00117 putative 40S ribosomal protein weak inendosperm PR00122 chlorophyll a/lo-binding protein very weak in shootprecursor (Cab27) PR00123 putative protochlorophyllide Strong leavesreductase PR00126 metallothionein RiCMT strong discrimination centershoot meristem PR00129 GOS2 Strong constitutive PR00131 GOS9 PR00133chitinase Cht-3 very weak meristem specific PR00135 alpha-globulinStrong in endosperm PR00136 alanine aminotransferase Weak in endospermPR00138 Cyclin A2 PR00139 Cyclin D2 PR00140 Cyclin D3 PR00141Cyclophyllin 2 Shoot and seed PR00146 sucrose synthase SS1 (barley)medium constitutive PR00147 trypsin inhibitor ITR1 (barley) weak inendosperm PR00149 ubiquitine 2 with intron strong constitutive PR00151WSI18 Embryo and stress PR00156 HVA22 homologue (putative) PR00157 EL2PR00169 aquaporine medium constitutive in young plants PR00170 Highmobility group protein Strong constitutive PR00171 reversiblyglycosylated protein weak constitutive RGP1 PR00173 cytosolic MDH shootPR00175 RAB21 Embryo and stress PR00176 CDPK7 PR00177 Cdc2-1 very weakin meristem PR00197 sucrose synthase 3 PRO0198 OsVP1 PRO0200 OSH1 veryweak in young plant meristem PRO0208 putative chlorophyllase PRO0210OsNRT1 PRO0211 EXP3 PRO0216 phosphate transporter OjPT1 PRO0218 oleosin18 kd aleurone + embryo PRO0219 ubiquitine 2 without intron PRO0220 RFLPRO0221 maize UBI delta intron not detected PRO0223 glutelin-1 PRO0224fragment of prolamin RP6 promoter PRO0225 4xABRE PRO0226 glutelinOSGLUA3 PRO0227 BLZ-2_short (barley) PR00228 BLZ-2_long (barley)

The nucleic acid construct of some embodiments of the invention canfurther include an appropriate selectable marker and/or an origin ofreplication. According to some embodiments of the invention, the nucleicacid construct utilized is a shuttle vector, which can propagate both inE. coli (wherein the construct comprises an appropriate selectablemarker and origin of replication) and be compatible with propagation incells. The construct according to the present invention can be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome.

The nucleic acid construct of some embodiments of the invention can beutilized to stably or transiently transform plant cells. In stabletransformation, the nucleic acid is integrated into the plant genome andas such it represents a stable and inherited trait. In transienttransformation, the exogenous polynucleotide is expressed by the celltransformed but it is not integrated into the genome and as such itrepresents a transient trait.

Thus, according to some aspects of the present invention, there isprovided an isolated cell comprising the nucleic acid construct of theinvention.

As used herein, the term “isolated cell” refers to a cell at leastpartially separated from the natural environment e.g., from a plant. Insome embodiments, the isolated cell is a plant cell of a whole plant. Insome embodiments, the isolated cell is a plant cell, for example, aplant cell in culture.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores. Plants that are particularly useful in the methods of theinvention include all plants which belong to the superfamilyViridiplantae, in particular monocotyledonous and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa,Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp,Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, maize,wheat, barley, rye, oat, peanut, pea, lentil and alfalfa, cotton,rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, atree, an ornamental plant, a perennial grass and a forage crop.Alternatively algae and other non-Viridiplantae can be used for themethods of the present invention.

According to some embodiments of the invention, the plant or plant cellis a duckweed plant, cell or nodule. Duckweed (members of themonocotyledonous family Lemnaceae, or Lemna) plant or duckweed nodulecultures can be efficiently transformed with an expression cassettecontaining a nucleotide sequence of interest by any one of a number ofmethods including Agrobacterium-mediated gene transfer, ballisticbombardment, or electroporation. Methods for molecular engineering ofduckweed cells and detailed description of duckweed expression systemsuseful for commercial production of polypeptides are known in the art(see, for example, U.S. Pat. Nos. 6,040,498 and 6,815,184 to Stomp, etal, and U.S. Pat. No. 8,022,270 to Dickey et al, all of which areincorporated fully by reference herein).

According to some embodiments of the invention, the plant or plant cellused by the method of the invention is a crop plant or cell of a cropplant such as rice, maize, wheat, barley, peanut, potato, sesame, olivetree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa,millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco,poplar and cotton.

According to further embodiments the plant cells includes tobacco cells,Agrobacterium rhizogenes transformed root cell, celery cell, gingercell, horseradish cell and carrot cells. In one embodiment the tobaccocells are from a tobacco cell line, such as, but not limited toNicotiana tabacum L. cv Bright Yellow (BY-2) cells. The plant cells maybe grown according to any type of suitable culturing method, includingbut not limited to, culture on a solid surface (such as a plasticculturing vessel or plate for example) or in suspension. It will benoted that some cells, such as the BY-2 and carrot cells can be culturedand grown in suspension. Suitable devices and methods for culturingplant cells in suspension are known in the art, for example, asdescribed in International Patent Application PCT IL2008/000614. In yetanother embodiment the cells are cells of whole tobacco plants or planttissues, including, but not limited to Nicotiana benthamiana.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. See, e.g., Horsch et al. in Plant Molecular BiologyManual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. Asupplementary approach employs the Agrobacterium delivery system incombination with vacuum infiltration. The Agrobacterium system isespecially viable in the creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

According to some embodiments of the invention, the transgenic plantsare generated by transient transformation of leaf cells, meristematiccells or the whole plant.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus(BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation ofplants using plant viruses is described in U.S. Pat. No. 4,855,237 (beangolden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese PublishedApplication No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); andGluzman, Y. et al., Communications in Molecular Biology: Viral Vectors,Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirusparticles for use in expressing foreign DNA in many hosts, includingplants are described in WO 87/06261.

According to some embodiments of the invention, the virus used fortransient transformations is avirulent and thus is incapable of causingsevere symptoms such as reduced growth rate, mosaic, ring spots, leafroll, yellowing, streaking, pox formation, tumor formation and pitting.A suitable avirulent virus may be a naturally occurring avirulent virusor an artificially attenuated virus. Virus attenuation may be effectedby using methods well known in the art including, but not limited to,sub-lethal heating, chemical treatment or by directed mutagenesistechniques such as described, for example, by Kurihara and Watanabe(Molecular Plant Pathology 4:259-269, 2003), Gal-on et al. (1992),Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression ofnon-viral nucleic acid sequences in plants is demonstrated by the abovereferences as well as by Dawson, W. O. et al., Virology (1989)172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al.Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990)269:73-76; and U.S. Pat. No. 5,316,931.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

In one embodiment, a plant viral polynucleotide is provided in which thenative coat protein coding sequence has been deleted from a viralpolynucleotide, a non-native plant viral coat protein coding sequenceand a non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral polynucleotide, andensuring a systemic infection of the host by the recombinant plant viralpolynucleotide, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native polynucleotidesequence within it, such that a protein is produced. The recombinantplant viral polynucleotide may contain one or more additional non-nativesubgenomic promoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or polynucleotide sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) polynucleotidesequences may be inserted adjacent the native plant viral subgenomicpromoter or the native and a non-native plant viral subgenomic promotersif more than one polynucleotide sequence is included. The non-nativepolynucleotide sequences are transcribed or expressed in the host plantunder control of the subgenomic promoter to produce the desiredproducts.

In a second embodiment, a recombinant plant viral polynucleotide isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral polynucleotide isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral polynucleotide. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native polynucleotidesequences may be inserted adjacent the non-native subgenomic plant viralpromoters such that the sequences are transcribed or expressed in thehost plant under control of the subgenomic promoters to produce thedesired product.

In a fourth embodiment, a recombinant plant viral polynucleotide isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral polynucleotide to produce a recombinant plantvirus. The recombinant plant viral polynucleotide or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral polynucleotide is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(exogenous polynucleotide) in the host to produce the desired protein.

Techniques for inoculation of viruses to plants may be found in Fosterand Taylor, eds. “Plant Virology Protocols: From Virus Isolation toTransgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods inVirology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A.“Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A.“Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa,eds. “Principles and Techniques in Plant Virology”, VanNostrand-Reinhold, New York.

In addition to the above, the polynucleotide of the present inventioncan also be introduced into a chloroplast genome thereby enablingchloroplast expression.

A technique for introducing exogenous nucleic acid sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous polynucleotide is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous polynucleotidemolecule into the chloroplasts. The exogenous polynucleotides selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the nucleic acid sequence includes, inaddition to a gene of interest, at least one polynucleotide stretchwhich is derived from the chloroplast's genome. In addition, theexogenous polynucleotide includes a selectable marker, which serves bysequential selection procedures to ascertain that all or substantiallyall of the copies of the chloroplast genomes following such selectionwill include the exogenous polynucleotide. Further details relating tothis technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507which are incorporated herein by reference. A polypeptide can thus beproduced by the protein expression system of the chloroplast and becomeintegrated into the chloroplast's inner membrane.

According to some embodiments of the invention, the method furthercomprises growing the plant cell expressing the nucleic acid. The plantcells can be any plant cells desired. The plant cells can be culturedcells, cells in cultured tissue or cultured organs, or cells in a plant.In some embodiments, the plant cells are cultured cells, or cells incultured tissue or cultured organs. In yet further embodiments, theplant cells are any type of plant that is used in gene transference. Theplant cell can be grown as part of a whole plant, or, alternatively, inplant cell culture.

According to some aspects of the invention, the plant cells are grown ina plant cell suspension culture. As used herein, the term “suspensionculture” refers to the growth of cells separate from the organism.Suspension culture can be facilitated via use of a liquid medium (a“suspension medium”). Suspension culture can refer to the growth ofcells in liquid nutrient media. Methods and devices suitable for growingplant cells of the invention in plant cell suspension culture aredescribed in detail in, for example, PCT WO2008/135991, U.S. Pat. No.6,391,683, U.S. patent application Ser. No. 10/784,295; InternationalPatent Publications PCT Nos.WO2004/091475, WO2005/080544 and WO2006/040761, all of which are hereby incorporated by reference as iffully set forth herein.

Thus, the invention encompasses plants or plant cultures expressing thenucleic acid sequences, so as to produce the recombinant chimericpolypeptide of the invention. Once expressed within the plant cell orthe entire plant, the level of the chimeric polypeptide encoded by thenucleic acid sequence can be determined by methods well known in the artsuch as, activity assays, Western blots using antibodies capable ofspecifically binding the chimeric polypeptide (anti TNFR2, and anti Fc,See Examples section which follows), Enzyme-Linked Immuno Sorbent Assay(ELISA), radio-immuno-assays (RIA), immunohistochemistry,immunocytochemistry, immunofluorescence and the like.

Methods of determining the level in the plant of the RNA transcribedfrom the nucleic acid sequence are well known in the art and include,for example, Northern blot analysis, reverse transcription polymerasechain reaction (RT-PCR) analysis (including quantitative,semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization.

According to some embodiments of the invention, the expressedrecombinant chimeric polypeptide of the present invention isglycosylated in the plant cell, resulting in a chimeric polypeptidehaving one, or two or three or more glycan structures having plantspecific glycan residues. Thus, according to some embodiments of theinvention, the cells expressing the expression vector of the inventionproduce a chimeric polypeptide having various amounts of glycanstructures arranged in one, two, three or more antennae. All structuresmay contain a core structure of two GlcNAcs and one mannose, andvariations of different amounts of mannose, in addition to core alpha(1,3) fucose, beta (1,2) xylose, and/or GlcNAc residues. Structures canbe of the high mannose type, having at least one, optionally at leasttwo, optionally at least three or optionally at least four or moremannose residues in addition to the core structure; or complex typehaving both mannose and other glycan types on each glycan, or of thehybrid type having both high mannose and complex antennae.

In other embodiments the cells expressing the expression vector of theinvention produce a chimeric polypeptide having at least one, optionallyat least two, optionally at least three or optionally at least four ormore core xylose residues. In yet other embodiments the cells expressingthe expression vector of the invention produce a chimeric polypeptidehaving at least one, optionally at least two, optionally at least threeor optionally at least four or more core α-(1,3) fucose residues. In oneembodiment the cells expressing the expression vector of the inventionproduce a chimeric polypeptide protein having at least one exposedmannose residue, at least one core xylose residue and at least oneα-(1,3) fucose residue. In yet further embodiments, the cells expressingthe expression vector of the invention produce a chimeric polypeptidehaving at least one, at least two, at least 3 or more terminal N-acetylglucosamine substitutions on the outer mannose sugars.

According to a specific embodiment the chimeric polypeptide lacks sialicacid residues. Yet further according to a specific embodiment, thechimeric polypeptide comprises at least 40%, 45%, 50%, 55%, 60%, 65%,70% or more complex glycans. According to a specific embodiment, thechimeric polypeptide comprises 40-70% complex glycans.

Purification of the secreted plant cell-expressed human chimericpolypeptide from the cell yields a highly purified compositioncomprising the prhTNFR2:Fc (also referred to herein as TNFR2:Fc orPRX-106). Thus, in some embodiments the chimeric polypeptide protein ispurified to a homogeneity of at least 98%. Thus the purified preparationis characterized by a purity of at least 85%, at least 87%, at least90%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, atleast 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%,at least 93.9%, at least 94%, at least 94.5%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,at least 99.7%, at least 99.8%, at least 99.9%, in a range of at least95.0-99.8% or 100% purity. In some embodiments, purity of the chimericpolypeptide is measured by HPLC.

In some embodiments the plant-expressed chimeric polypeptide preparationcomprises impurities derived from the plant host cell, such as, but notlimited to nucleic acids and polynucleotides, amino acids, oligopeptidesand polypeptides, glycans and other carbohydrates, lipids and the like.In some embodiments the host-cell derived impurities comprisebiologically active molecules, such as enzymes. In other embodiments,the plant-expressed chimeric polypeptide composition comprises plantbeta-N-acetylhexosaminidase. Where the host cell is a tobacco cell, ortobacco cell line cell, the plant beta-N-acetylhexosaminidase is atobacco beta-N-acetylhexosaminidase.

In further embodiments the plant beta-N-acetylhexosaminidase isinactivated plant beta-N-acetylhexosaminidase. Inactivation of plantbeta-N-acetylhexosaminidase can be effected by physical means, chemicalmeans or biochemical means. Physical inactivation can be performed byheating, freezing, desiccation, etc. Chemical inactivation can beperformed by extremes of pH, chemical denaturation, addition or removalof side chains, glycans, amino acids, etc. Biochemical inactivationincludes, but is not limited to inhibition by reversible or irreversibleinhibitors. Exemplary beta-N-acetylhexosaminidase inhibitors includeend-product inhibitors such as N-acetyl-D-glucosamine andbeta-methyl-N-acetyl glucosamine, and selective inhibitors such as thecompounds disclosed in US Patent Applications US2010016386,US20110237631, US20100087477 and US20120046337. It will be appreciatedthat preferred methods for inhibition and/or inactivation of the plantbeta-N-acetylhexosaminidase are those which also effectively preservethe structural and functional integrity of the plant-expressed chimericpolypeptide.

In some embodiments the plant beta-N-acetylhexosaminidase is inactivatedby heating the composition comprising the chimeric polypeptide. Suitabletemperatures for plant beta-N-acetylhexosaminidase inhibition and/oractivation include heating within a range of 37-60° C. for a period of 2to 5, 10, 20, 30, 40, 50, 60 or more minutes. It will be appreciatedthat effective inhibition and/or inactivation of the plantbeta-N-acetylhexosaminidase is achieved more rapidly at highertemperatures and more slowly at lower temperatures of the range. In someembodiments, the plant-expressed chimeric polypeptide composition isheated in the range of 45-55° C. for 2-10 minutes. In some embodiments,the inhibition/inactivation results in 20, 30, 40, 50, 60, 70, 80% orgreater inactivation of the plant beta-N-acetylhexosaminidase.

The chimeric polypeptide of the invention is utilized for the treatmentof TNFα-associated medical conditions.

The term “treating” refers to inhibiting, preventing or arresting thedevelopment of a pathology (disease, disorder or condition) and/orcausing the reduction, remission, or regression of a pathology. Those ofskill in the art will understand that various methodologies and assayscan be used to assess the development of a pathology, and similarly,various methodologies and assays may be used to assess the reduction,remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease,disorder or condition from occurring in a subject who may be at risk forthe disease, but has not yet been diagnosed as having the disease.

As used herein “a TNFα-associated medical condition” refers to a medicalcondition in which TNFα activity is associated with onset, progressionof the medical conditions and/or related symptoms in a subject.

Thus, TNFα-associated medical condition disease, in a cell, tissue,organ, animal, or subject in need thereof including, but not limited to,at least one of obesity, an immune related disease, a cardiovasculardisease, an infectious disease, a malignant disease or a neurologicdisease (see WO0212502).

Specific examples of a TNFα-associated medical condition include, butare not limited to, rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn'sdisease, inflammatory bowel disease, short bowel syndrome, cholitis,ulcerative cholitis, chronic obstructive pulmonary disease (COPD),Hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopicdermatitis. Additional diseases or disorders that can be treated withthe chimeric polypeptide of the invention include those described in WO00/62790, WO 01/62272 and U.S. Patent Application No. 2001/0021380, U.S.Pat. No. 7,648,702, the relevant portions of which are incorporatedherein by reference.

Other examples of TNFα-associated medical conditions include, but arenot limited to those disclosed in Kuek et al. Postgrad. Med. J. 2007;83; 251-260, which is herein incorporated by reference in its entirety.

Thus, exemplary indications include, Sjorgen's syndrome, polymyositis,dermatomyositis, Wegener's vasculitis, Bechet's, giant cell arteritis(GCA), Polymyalgia rheumatic, Takayasu's arteritis, Polyarteritisnodosa, Sarcoidosis, adult onset Still's disease, Kawasaki disease,Cryoglobulinemic vasculitis, relapsing polychondritis, Hidradenitissuppurativa, Coeliac disease, myelodysplastic syndromes, Pyodermagangrenosum, Erythema nodosum, SAPHO syndrome, graft versus hostdisease, chronic hepatitis B/C, thrombic/idiopathic thrombocytopenicpurpura, refractory asthma, lupus, amyloidosis, Multicentricreticulohistiocytosis, pemphigus, Grave's disease, antiphospholipidsyndrome, idiopathic membranous glomerulonephritis, Hep C associatedglomerulonephritis, myasthenia gravis and multiple sclerosis.

According to a specific embodiment, TNFα-associated medical condition isan inflammatory bowel disease. According to a specific embodiment, thepolypeptide is administered enterally, e.g., orally, such as comprisedin the plant cells.

According to a specific embodiment, the inflammatory bowel disease isulcerative colitis or Crohn's disease. According to a specificembodiment, the polypeptide is administered enterally, e.g., orally,such as comprised in the plant cells.

Additional examples of TNFα-associated medical condition include, butare not limited to, immune related disease, such as rheumatoidarthritis, juvenile rheumatoid arthritis, systemic onset juvenilerheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis,gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatorybowel disease, short bowel syndrome, ulcerative colitis, systemic lupuserythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/opticneuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolytic disease,thrombocytopenia, graft rejection of any organ or tissue, kidneytranslplant rejection, heart transplant rejection, liver transplantrejection, pancreas transplant rejection, lung transplant rejection,bone marrow transplant (BMT) rejection, skin allograft rejection,cartilage transplant rejection, bone graft rejection, small boweltransplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions-, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), polyneuropathy, organomegaly,endocrinopathy, monoclonal gammopathy, skin changes syndrome,antiphospholipid syndrome, pemphigus, scleroderma, mixed connectivetissue disease, idiopathic Addison's disease, diabetes mellitus, chronicactive hepatitis, primary billiary cirrhosis, vitiligo, vasculitis,post-MI cardiotomy syndrome, type IV hypersensitivity, contactdermatitis, hypersensitivity pneumonitis, allograft rejection,granulomas due to intracellular organisms, drug sensitivity,metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g, including but not limited toasthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g, theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al,eds. Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference. The present invention alsoprovides a method for treating at least one cardiovascular disease in acell, tissue, organ, animal, or patient, including, but not limited to,at least one of cardiac stun syndrome, myocardial infarction, congestiveheart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis,atherosclerosis, restenosis, diabetic ateriosclerotic disease,hypertension, arterial hypertension, renovascular hypertension, syncope,shock, syphilis of the cardiovascular system, heart failure, corpulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrialectopic beats, atrial flutter, atrial fibrillation (sustained orparoxysmal), post perfusion syndrome, cardiopulmonary bypassinflammation response, chaotic or multifocal atrial tachycardia, regularnarrow QRS tachycardia, specific arrythmias, ventricular fibrillation,His bundle arrythmias, atrioventricular block, bundle branch block,myocardial ischemic disorders, coronary artery disease, angina pectoris,myocardial infarction, cardiomyopathy, dilated congestivecardiomyopathy, restrictive cardiomyopathy, valvular heart diseases,endocarditis, pericardial disease, cardiac tumors, aordic and peripheralaneuryisms, aortic dissection, inflammation of the aorta, occulsion ofthe abdominal aorta and its branches, peripheral vascular disorders,occulsive arterial disorders, peripheral atherlosclerotic disease,thromboangitis obliterans, functional peripheral arterial disorders,Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venousdiseases, venous thrombosis, varicose veins, arteriovenous fistula,lymphederma, lipedema, unstable angina, reperfusion injury, post pumpsyndrome, ischemia-reperfusion injury, and the like.

Additional examples of a TNFα-associated medical condition include, butare not limited to, infectious diseases, such as acute or chronicbacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis (A, B or C, or thelike), septic arthritis, peritonitis, pneumonia, epiglottitis, e. coli0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like.

Additional examples of a TNFα-associated medical condition include, butare not limited to, malignant diseases such as leukemia, acute leukemia,acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acutemyeloid leukemia (AML), chronic myelocytic leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome(MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas,sarcomas, malignant melanoma, hemangioma, metastatic disease, cancerrelated bone resorption, cancer related bone pain, and the like.

Additional examples of a TNFα-associated medical condition include, butare not limited to, neurologic diseases, such as neurodegenerativediseases, multiple sclerosis, migraine headache, AIDS dementia complex,demyelinating diseases, such as multiple sclerosis and acute transversemyelitis; extrapyramidal and cerebellar disorders' such as lesions ofthe corticospinal system; disorders of the basal ganglia or cerebellardisorders; hyperkinetic movement disorders such as Huntington's Choreaand senile chorea; drug-induced movement disorders, such as thoseinduced by drugs which block CNS dopamine receptors; hypokineticmovement disorders, such as Parkinson's disease; Progressive supranucleoPalsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multisystem disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit, such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyofrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like.

As used herein, the term “subject” includes mammals, e.g., human beingsat any age which suffer from the pathology. According to a specificembodiment, this term encompasses individuals who are at risk to developthe pathology.

It has been shown that plant cells expressing biologically active humanrecombinant polypeptides can be used as an effective systemic deliverysystem, when provided for enteral administration to the subject (seeWO2007/010533). Thus, in some embodiments, the chimeric polypeptide canbe formulated in a pharmaceutical composition for oral or enteraldelivery comprising transformed plant cell expressing the chimericpolypeptide and a pharmaceutically acceptable carrier. In someembodiments, the transformed plant cells of the pharmaceuticalcomposition are lyophilized plant cells.

As used herein the phrase “enteral administration” refers toadministration through any part of the gastro-intestinal tract, such asrectal administration, colonic administration, intestinal administration(proximal or distal) and gastric administration. In some embodiments,enteral administration refers to oral administration.

It will be appreciated that the present teachings are also directed atmucosal administration of plant cells expressing the chimericpolypeptide of the invention.

The cells may be formulated as a solid, formulated as a liquid orformulated as a powder. In some embodiments, the cells are resuspended,lyophilized cells.

Thus, in some embodiments, the chimeric polypeptide can be formulated ina pharmaceutical composition for oral or enteral delivery comprisingtransformed plant cell expressing the chimeric polypeptide and apharmaceutically acceptable carrier. In some embodiments, thetransformed plant cells of the pharmaceutical composition arelyophilized plant cells, although the use of fresh (non-lyophilizedcells), plant tissues, plant parts or whole plants is also contemplatedherein.

Prior to lyophilization the cells may be washed to remove any celldebris that may be present in the growth medium.

As the cells are being prepared for lyophilization, it is sometimesdesirable to incubate the cells in a maintenance medium to reduce themetabolic processes of the cells.

Pretreatment (although not necessary) can be performed at roomtemperature or at temperatures in which the plant cells are typicallycultured. Pretreatment is performed at about room temperature (20° C.)for ease of handling and as most plant cells are fairly stable at roomtemperature. Stabilizers can be added directly to the medium andreplenished as necessary during the pretreatment process.

Pretreatments may also involve incubating cells in the presence of oneor more osmotic agents. Examples of useful osmotic agents include sugarssuch as saccharides and saccharide derivatives, amino or imino acidssuch as proline and proline derivatives, or combinations of theseagents. Some of the more useful sugars and sugar derivatives arefructose, glucose, maltose, mannitol, sorbitol, sucrose and trehalose.Osmotic agents are utilized at a concentration that prepares cells forsubsequent lyophilization.

Lyophilization is directed at reducing the water content of the cells byvacuum evaporation. Vacuum evaporation involves placing the cells in anenvironment with reduced air pressure. Depending on the rate of waterremoval desired, the reduced ambient pressure operating at temperaturesof between about −30° C. to −50° C. may be at 100 torr, 1 torr, 0.01torr or less. According to a specific embodiment, the cells arelyophilized by freezing to −40° C. and then applying a vacuum to apressure of 0.1 mbar. The cells are then heated to −10° C. so all theice content will be sublimated and evaporated. Under conditions ofreduced pressure, the rate of water evaporation is increased such thatup to 60-95% of the water in a cell can be removed.

According to a specific embodiment, lyophilization removes over 60%,70%, 80% or specifically over 90%, 91%, 92%, 93%, 94%, 95% or 98% of thewater from the cells. According to a specific embodiment, the finalwater content is about 5-10%, 5-8% or 6-7%.

Thus, the oral dosage form may be provided as an oral nutritional form(e.g., as long as the protein is not exposed to denaturing conditionswhich include heating above 37° C. and compression), as a complete meal,as a powder for dissolution, e.g. health drinks, as a solution, as aready-made drink, optionally low calorie, such as a soft drink,including juices, milk-shake, yoghurt drink, smoothie or soy-baseddrink, in a bar, or dispersed in foods of any sort, such as bakedproducts, cereal bars, dairy bars, snack-foods, breakfast cereals,muesli, candies, tabs, cookies, biscuits, crackers (such as a ricecrackers), chocolate, and dairy products.

Of note is the use of plant cells expressing the chimeric polypeptide inthe treatment of inflammatory bowel disease. The plant's cell wall isexpected to protect the chimeric polypeptide while moving through thestomach and small intestine. In the colon, where the polysaccharides aredigested, the plant cell is expected to release its content and henceprh TNRF:Fc is available to bind its cytokine ligand. Moreover, prhTNRF2:Fc is a chimeric protein carries an Fc segment of human IgG1. Inthe epithelial monolayer lining the mucosal barrier, the FcRn receptortranscytoses IgG molecules across by binding to their Fc. Therefore, prhTNRF:Fc can also cross the epithelial barrier to bind its cytokineligand on the serosal side of the epithelia.

It will be appreciated that the present teachings exclude the use ofplant cells expressing the chimeric polypeptide by enteraladministration for the treatment of medical conditions directlyassociated with obesity, metabolic syndrome, diabetes and a liverdisease or disorder.

The metabolic syndrome is a constellation of interrelated risk factorsof metabolic origin—metabolic risk factors—that appear to directlypromote the development of atherosclerotic cardiovascular disease(ASCVD). Patients with the metabolic syndrome also are at increased riskfor developing type 2 diabetes mellitus. The multiple components andcriteria that define the metabolic syndrome have varied somewhat inspecific elements, but in general they include a combination of bothunderlying and metabolic risk factors. The most widely recognized of themetabolic risk factors are atherogenic dyslipidemia, elevated bloodpressure, and elevated plasma glucose. Individuals with thesecharacteristics commonly manifest a prothrombotic state and aproinflammatory state as well. Atherogenic dyslipidemia consists of anaggregation of lipoprotein abnormalities including elevated serumtriglyceride and apolipoprotein B (apoB), increased small LDL particles,and a reduced level of HDL cholesterol (HDL-C). Available data suggestthat it truly is a syndrome, i.e., a grouping of ASCVD risk factors, butone that probably has more than one cause.

According to a specific embodiment, the liver disease or disorder isselected from the group consisting of hepatitis, liver cirrhosis, livercancer, hepatotoxicity, chronic liver disease, fatty liver disease andnon-alcoholic steatohepatitis (NASH).

According to a specific embodiment, when the TNFR2:Fc is administeredenterally (e.g., orally) in plant cells, the medical condition is not anobesity, metabolic syndrome, diabetes and a liver disease or disorder.

According to a specific embodiment, the hepatotoxicity is induced by achemical agent selected from the group consisting of acetaminophen,NSAIDS, glucocorticoid, isoniazid, arsenic, chemotherapy, carbontetrachloride and vinyl chloride.

According to a specific embodiment, the diabetes is selected from thegroup consisting of type I diabetes, type II diabetes and LADA disease.

Alternatively or additionally, the chimeric polypeptide of someembodiments of the invention can be administered to an organism per se,or in a pharmaceutical composition where it is mixed with suitablecarriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the chimeric polypeptideor cells expressing same accountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, intraperitoneal, intranasal, orintraocular injections.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, and/or intrathecal. Parenteraladministration can be by bolus injection or continuous infusion.Pharmaceutical compositions for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-Ease™, Genject™, injector pens such as GenPen™,and needleless devices such as MediJector™ and BioJector™.

The pharmaceutical composition can also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions of some embodiments of the invention may bemanufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodimentsof the invention thus may be formulated in conventional manner using oneor more physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to some embodiments of the invention are convenientlydelivered in the form of an aerosol spray presentation from apressurized pack or a nebulizer with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of some embodiments of the invention mayalso be formulated in rectal compositions such as suppositories orretention enemas, using, e.g., conventional suppository bases such ascocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of someembodiments of the invention include compositions wherein the activeingredients are contained in an amount effective to achieve the intendedpurpose. More specifically, a therapeutically effective amount means anamount of active ingredients (chimeric polypeptide) effective toprevent, alleviate or ameliorate symptoms of a disorder or prolong thesurvival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide thechimeric polypeptide (the target tissue) levels of the active ingredientare sufficient to induce or suppress the biological effect (minimaleffective concentration, MEC). The MEC will vary for each preparation,but can be estimated from in vitro data. Dosages necessary to achievethe MEC will depend on individual characteristics and route ofadministration. Detection assays can be used to determine plasmaconcentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

In one embodiment, the effective chimeric polypeptide amount per adultdose ranges from about 1-500 mg/m², or from about 1-200 mg/m², or fromabout 1-40 mg/m² or about 5-25 mg/m². Alternatively, a flat dose may beadministered, whose amount may range from 2-500 mg/dose, 2-100 mg/doseor from about 10-80 mg/dose.

In one embodiment, the effective chimeric polypeptide amount per adultdose is about 1-500 mg/m², or about 1-200 mg/m², or about 1-40 mg/m² orabout 5-25 mg/m². Alternatively, a flat dose may be administered, whoseamount may range about 2-500 mg/dose, 2-100 mg/dose or from about 10-80mg/dose.

In another embodiment the effective chimeric polypeptide amount peradult dose range is about 0.0002 mg/kg to 2 mg/kg, about 0.002-2 mg/kg,about 0.02-2 mg/kg, about 0.2-2 mg/kg, about 0.002-0.2 mg/kg, about0.0002-1 mg/kg, about 0.002-0.1 mg/kg, about 0.002-0.02 mg/kg, about0.002-0.01 mg/kg, about 0.002-0.008 mg/kg, about 0.02-0.1 mg/kg, about0.001-0.05 mg/kg, about 0.001-0.01 mg/kg, about 0.01-1 mg/kg, about0.01-15 mg/kg, about 0.005-1 mg/kg, about 0.01-5 mg/kg, about 0.005-0.01mg/kg or about 0.05-0.1 mg/kg. According to a further specificembodiment, the effective chimeric polypeptide amount per adult doserange is about 0.02-0.3 mg/kg, 0.02-0.7 mg/kg, 0.02-0.247 mg/kg,0.02-0.12 mg/kg, 0.12-0.24 mg/kg, 0.02-0.1 mg/kg or 0.15-0.247 mg/kg.

According to a specific embodiment, these dose ranges are used for oraladministration such as of plant cells expressing the chimeric protein.

According to a specific embodiment, the effective chimeric polypeptideamount per adult dose ranges about 0.002-0.2 mg/kg or 0.02-0.3 mg/kg.According to a specific embodiment the effective chimeric polypeptideamount per adult dose ranges about 0.02-0.3 mg/kg, 0.02-0.27 mg/kg or0.02-0.247 mg/kg. According to a specific embodiment, this dose range isused for oral administration such as of plant cells expressing thechimeric protein.

Alternatively, a flat dose may be administered, whose amount may rangeabout 2-500 mg/dose, 2-100 mg/dose or from 10-80 mg/dose. According to aspecific embodiment, this dose range is used for oral administrationsuch as of plant cells expressing the chimeric protein.

According to a specific embodiment, a flat dose of 0.01-100 mg, 0.1-100mg, 0.1-50 mg, 1-20 mg, 1-10 mg, 10-20 mg, 15-20 mg, 0.1-20 mg, 0.1-10mg, 0.1-5 mg is administered. According to a specific embodiment, thisdose range is used for oral administration such as of plant cellsexpressing the chimeric protein.

Any of these doses can be packed in a unit dosage form which comprisesplant cells expressing (per batch and not per cell) the polypeptide inan amount of 1-20 mg, 1-10 mg, 10-20 mg or 15-20 mg.

According to a specific embodiment the flat dose is about 0.1-10 mg.According to a specific embodiment, this dose range is used for oraladministration such as of plant cells expressing the chimeric protein.

According to a specific embodiment, the oral dose is administered daily.The dose may be divided for a number of administrations during the day(say 2-4 times a day). The dose can also be administered every two days,two times a week, three times a week, biweekly, weekly doses, orseparated by several weeks (for example 2 to 8).

According to a specific embodiment, if the dose is to be administeredmore than one time per week, an exemplary dose range is the same as theforegoing described dose ranges or lower and administered two or moretimes per week (e.g., 25-100 mg/dose). In another embodiment, anacceptable dose for administration by injection contains 80-100 mg/dose,or alternatively, containing 80 mg per dose.

The dose may be modified for children and infants.

The dose can be administered at biweekly, weekly doses, or separated byseveral weeks (for example 2 to 8). According to a specific embodimentthe chimeric polypeptide is generally administered at 25 mg by a singlesubcutaneous (SC) injection.

In many instances, an improvement in a patient's condition will beobtained by a dose of up to about 100 mg of the pharmaceuticalcomposition one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For incurable chronic conditions theregimen may be continued indefinitely. For pediatric patients (ages4-17), a suitable regimen involves a dose of 0.4 mg/kg to 5 mg/kg of thechimeric polypeptides of the invention by injection, administered one ormore times per week.

In another embodiment, it is contemplated that the pharmaceuticalformulation of the invention is prepared in a bulk formulation and assuch, the components of the pharmaceutical composition are adjusted sothat it is higher than would be required for administration and dilutedappropriately prior to administration.

Compositions of some embodiments of the invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions comprising a preparation of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition, as is further detailed above. Theconcentration of the polypeptide in the aqueous pharmaceuticalcomposition can vary over a wide range, but is generally within therange of from about 0.05 to about 20,000 micrograms per milliliter μ/ml)of aqueous formulation.

Of note dosage forms which comprise the plant cells may includeadditives such as one or more of calcium, magnesium, iron, zinc,phosphorus, vitamin D and vitamin K. A suitable daily amount is 0.1 mgto 3.6 g calcium, preferably 320 to 530 mg. In general, the daily dosageof vitamins and minerals in the nutritional formulation or medicament ofthe invention is 25-100% by weight of the dosages recommended by thehealth authorities. Dietary fiber may also be a component of thecompositions of the invention. Further components of the supplement mayinclude any bioactive compounds or extracts which are known to havehealth benefits, especially for improving physical performance.

Generally the unit dosage form may further comprise an antioxidant(exemplary embodiments are provided above. In another embodiment, theantioxidant is a pharmaceutically acceptable antioxidant. In anotherembodiment, the antioxidant is selected from the group consisting ofvitamin E, superoxide dismutase (SOD), omega-3, and beta-carotene.

In another embodiment, the unit dosage form further comprises anenhancer of the biologically active protein or peptide. In anotherembodiment, the unit dosage form further comprises a cofactor of thebiologically active protein or peptide.

In another embodiment, a unit dosage form of the present inventionfurther comprises pharmaceutical-grade surfactant. Surfactants are wellknown in the art, and are described, inter alia, in the Handbook ofPharmaceutical Excipients (eds. Raymond C Rowe, Paul J Sheskey, and SianC Owen, copyright Pharmaceutical Press, 2005). In another embodiment,the surfactant is any other surfactant known in the art.

In another embodiment, a unit dosage form of the present inventionfurther comprises pharmaceutical-grade emulsifier or emulgator(emollient). Emulsifiers and emulgators are well known in the art, andare described, inter alia, in the Handbook of Pharmaceutical Excipients(ibid). Non-limiting examples of emulsifiers and emulgators areeumulgin, Eumulgin B1 PH, Eumulgin B2 PH, hydrogenated castor oilcetostearyl alcohol, and cetyl alcohol. In another embodiment, theemulsifier or emulgator is any other emulsifier or emulgator known inthe art.

In another embodiment, a unit dosage form of the present inventionfurther comprises pharmaceutical-grade stabilizer. Stabilizers are wellknown in the art, and are described, inter alia, in the Handbook ofPharmaceutical Excipients (ibid). In another embodiment, the stabilizeris any other stabilizer known in the art.

In another embodiment, a unit dosage form of the present inventionfurther comprises an amino acid selected from the group consisting ofarginine, lysine, aspartate, glutamate, and histidine. In anotherembodiment, analogues and modified versions of arginine, lysine,aspartate, glutamate and histidine are included in the terms “arginine,”“lysine,” “aspartate”, “glutamate” and “histidine,” respectively. Inanother embodiment, the amino acid provides additional protection ofribonuclease or other active molecules. In another embodiment, the aminoacid promotes interaction of biologically active protein or peptide witha target cell. In another embodiment, the amino acid is contained in anoil component of the unit dosage form.

In another embodiment, a unit dosage form of the present inventionfurther comprises one or more pharmaceutically acceptable excipients,into which the matrix carrier unit dosage form is mixed. In anotherembodiment, the excipients include one or more additionalpolysaccharides. In another embodiment, the excipients include one ormore waxes. In another embodiment, the excipients provide a desiredtaste to the unit dosage form. In another embodiment, the excipientsinfluence the drug consistency, and the final dosage form such as a gelcapsule or a hard gelatin capsule.

Non limiting examples of excipients include: Antifoaming agents(dimethicone, simethicone); Antimicrobial preservatives (benzalkoniumchloride, benzelthonium chloride, butylparaben, cetylpyridiniumchloride, chlorobutanol, chlorocresol, cresol, ethylparaben,methylparaben, methylparaben sodium, phenol, phenylethyl alcohol,phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate,potassium sorbate, propylparaben, propylparaben sodium, sodium benzoate,sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal,thymol); Chelating agents (edetate disodium, ethylenediaminetetraaceticacid and salts, edetic acid); Coating agents (sodiumcarboxymethyl-cellulose, cellulose acetate, cellulose acetate phthalate,ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,methacrylic acid copolymer, methylcellulose, polyethylene glycol,polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcrystalline wax, zein); Colorants (caramel, red,yellow, black or blends, ferric oxide); Complexing agents(ethylenediaminetetraacetic acid and salts (EDTA), edetic acid, gentisicacid ethanolmaide, oxyquinoline sulfate); Desiccants (calcium chloride,calcium sulfate, silicon dioxide); Emulsifying and/or solubilizingagents (acacia, cholesterol, diethanolamine (adjunct), glycerylmonostearate, lanolin alcohols, lecithin, mono- and di-glycerides,monoethanolamine (adjunct), oleic acid (adjunct), oleyl alcohol(stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl 35 casteroil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleyl ether,polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, propylene glycoldiacetate, propylene glycol monostearate, sodium lauryl sulfate, sodiumstearate, sorbitan monolaurate, sorbitan monooleate, sorbitanmonopalmitate, sorbitan monostearate, stearic acid, trolamine,emulsifying wax); Flavors and perfumes (anethole, benzaldehyde, ethylvanillin, menthol, methyl salicylate, monosodium glutamate, orangeflower oil, peppermint, peppermint oil, peppermint spirit, rose oil,stronger rose water, thymol, tolu balsam tincture, vanilla, vanillatincture, vanillin); Humectants (glycerin, hexylene glycol, propyleneglycol, sorbitol); Polymers (e.g., cellulose acetate, alkyl celluloses,hydroxyalkylcelluloses, acrylic polymers and copolymers); Suspendingand/or viscosity-increasing agents (acacia, agar, alginic acid, aluminummonostearate, bentonite, purified bentonite, magma bentonite, carbomer934p, carboxymethylcellulose calcium, carboxymethylcellulose sodium,carboxymethycellulose sodium 12, carrageenan, microcrystalline andcarboxymethylcellulose sodium cellulose, dextrin, gelatin, guar gum,hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin,polyethylene oxide, polyvinyl alcohol, povidone, propylene glycolalginate, silicon dioxide, colloidal silicon dioxide, sodium alginate,tragacanth, xanthan gum); Sweetening agents (aspartame, dextrates,dextrose, excipient dextrose, fructose, mannitol, saccharin, calciumsaccharin, sodium saccharin, sorbitol, solution sorbitol, sucrose,compressible sugar, confectioner's sugar, syrup). This list is not meantto be exclusive, but instead merely representative of the classes ofexcipients and the particular excipients which may be used in oraldosage unit dosage forms of the present invention.

Conventional additives may be included in the compositions of theinvention, including any of those selected from preservatives, chelatingagents, effervescing agents, natural or artificial sweeteners, flavoringagents, coloring agents, taste masking agents, acidulants, emulsifiers,thickening agents, suspending agents, dispersing or wetting agents,antioxidants, and the like. Flavoring agents can be added to thecompositions of the invention to aid in compliance with a dosingregimen. Typical flavoring agents include, but are not limited tonatural or synthetic essences, oils and/or extracts of pineapple,orange, lemon, mint, berry, chocolate, vanilla and melon.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Materials and Experimental Procedures

Expression Constructs and Expression

cDNA encoding prh TNFR2:Fc was optimized and synthesized by GENEART AG(Regensburg, Germany). The codon usage was adapted to the codon bias ofNicotiana tabacum genes. The IgG1 portion was cloned from Fc IgG1 heavychain constant region [Homo sapiens] ACCESSION AEV43323.

During the optimization process the following cis-acting sequence motifswere avoided: Internal TATA-boxes, chi-sites and ribosomal entry sites,AT-rich or GC-rich sequence stretches, RNA instability elements (“Killermotifs”), Repeat sequences and RNA secondary structures, splice donor(cryptic) and acceptor sites, branch points. In addition, regions ofvery high (>80%) or very low (<30%) GC content were avoided. Theresultant DNA sequence is as set forth in SEQ ID NO: 1. The encodedpolypeptide is as set forth in SEQ ID NO: 2. To the native cDNAsequence, a signal peptide (e.g. endoplasmic reticulum target signalpeptide) from N. plumbaginifolia Calreticulin protein was added to theN′ terminus of the gene, allowing efficient targeting of prh TNFR2:Fc tothe secretory pathway and is then cleaved from the polypeptide, bysignal peptidase, once the protein has been translocated into theendoplasmic reticulum (SEQ ID NO: 3, SEQ ID NO: 4, representing the DNAand peptide sequences of the ER signal peptide, respectively).Additionally, an ER retention signal SEKDEL was added to the C′ terminusof the gene. This signal allows protein retrieval from the Golgiapparatus to the ER, and localization in the ER. The entire codingsequence (signal peptide-prh TNFR2:Fc-SEKDEL) is as set forth in SEQ IDNO: 5 and the encoded polypeptide is as set forth in SEQ ID NO: 6. Theresultant protein following cleavage is as set forth in SEQ ID NO: 7,204 or 205 (prh TNFR2:Fc-SEKDEL).

Stable Expression in N. tabacum BY2 Cells

Agrobacterium mediated transformation is widely used to introduceforeign genes into a plant cell genome. Using this approach, a T-DNAmolecule consisting of a foreign gene and its regulatory elements israndomly introduced into the plant genome. Since the site ofintegration, as well as the copy number of the gene insertions cannot becontrolled, the transformation process results in a highly heterogeneoustransgenic ‘pool’ composed of cells with various levels of transgeneexpression. The transgenic ‘pool’ is subsequently used for cloneisolation. The transformation process, results in establishment ofnumerous single cell lines, each representing an individualtransformation event, from which the clone with the highest expressionlevel of the foreign gene is selected. For prh TNFR2:Fc, thetransformation was conducted with a plasmid carrying the prh TNFR2:Fccassette (FIG. 1 SEQ ID NOs: 7 and 8). As a result, the recombinantprotein is targeted to the Endoplasmic reticulum (ER) of the cells. Thetransformations of the BY2 cells with the prh TNFR2:FC-ER expressionvector were performed by the Agrobacterium tumefaciens mediated planttransformation procedure as follow: BY2 (Bright Yellow 2) suspensionculture was co-cultivated, for 48 hours, with the Agrobacteriumtumefactiens strain carrying the vector harboring the prhTNFR2:FC-geneand the neomycin phosphotransferase (NPTII) selection gene.Subsequently, cells were kept in media supplemented with 50 mg/L ofKanamaycin and 250 mg/L Cefotaxime. The NPTII gene confers resistance toKanamycin, thus only NPTII positive BY2 cells survive in this selectionmedia. The Cefotaxime was used to selectively kill the agrobacterium,the plant cells being resistant to this antibiotic.

Screening for the Optimal Expressing Clone

In order to select individual cell lines, aliquots of highly dilutedcell suspension were spread on solid BY-2 medium (Toshiyuki Nagata &Fumi Kumagai Methods in Cell Science 21: 123-127, 1999). The cells werethen grown until small calli developed. Each callus was thenre-suspended in liquid culture. Cells were then sampled and evaluatedfor prh TNFR2:FC. About 500 cell lines were screened by Western blotunder denaturing conditions (FIG. 4). The lines with high expressionlevels were further re-analyzed by the same method to select the highestexpressing clone of prh TNFR2:FC producing clone.

Gel Electrophoresis:

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)separates proteins on an electrical field according to their size.Proteins in the presence of the detergent SDS migrate as a linearfunction of the logarithm of their molecular weight. Migration patternand identification of prh TNFR2:FC on SDS-PAGE was compared tocommercial molecular weight standard proteins (New England BioLabs; catNo. P7708S) and to the commercially available, mammalian-cell derivedEnbrel expressed in CHO cells (Entanercept; Wyeth). prh TNFR2:FC wasextracted from cells either by reducing sample buffer containingβ-mercaptoethanol or by native extraction buffer. The native extractionsupernatant was mixed with non-reducing sample buffer prior to analysis.Electrophoresis was performed using Criterion™ cell verticalelectrophoresis apparatus (Bio-Rad Lab.) with premixed electrophoresisTris-Glycine-SDS running buffer (Bio-Rad Laboratories). Followingelectrophoresis, the proteins were transferred from the Polyacrylamidegel to a protein binding nitrocellulose membrane (iBlot™). Membraneswere blocked for 1 hr at RT with 5% milk buffer containing 0.1% Tween20. For identification of the Fc portion of the molecule, Goat antihuman IgG conjugated to HRP (cat #109-035-098, Jackson.) was used. ForTNFR2 detection, a Rabbit Anti-TNFRII (ID: ab109853, Abcam) followed byGoat anti Rabbit HRP (cat #111-035-003, Jackson) were employed.Detection was carried out with ECL detection kit (Pierce). Theimmunoreactivity of prh TNFR2:FC was compared to that of commercialEnbrel (Entanercept; Wyeth). Bands were detected using the MolecularImager Gel Doc XR System (Bio-Rad Laboratories).

Amino Acid Sequencing by Mass-Spectrometry

prhTNFR2:FC is sent for sequencing analysis at the Smoler ProteomicsCenter at the Technion—Israel Institute of Technology (Haifa, Israel).The protein is extracted from the gel, reduced with 2.8 mM DTT (60° C.for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammoniumbicarbonate (in the dark, room temperature for 30 min) and digested in10% ACN and 10 mM ammonium bicarbonate with modified Trypsin (Promega)or with ChymoTrypsin overnight at 37° C. in a 1:50 enzyme-to-substrateratio. 3% of the resulting peptides are resolved by reverse-phasechromatography on 0.075×200-mm fused silica capillaries (J&W) packedwith Reprosil reversed phase material (Dr Maisch GmbH, Germany). Thepeptides are eluted with linear 60 minutes gradients of 5 to 45% and 15minutes at 95% acetonitrile with 0.1% formic acid in water at flow ratesof 0.25 μmin. On line mass spectrometry is performed by an ion-trap massspectrometer (Orbitrap, Thermo) in a positive mode using repetitivelyfull MS scan followed by collision induces dissociation (CID) of the 7most dominant ion selected from the first MS scan.

The mass spectrometry data is analyzed using the Sequest 3.31 software(J. Eng and J. Yates, University of Washington and Finnigan, San Jose)vs a specific sequence.

Glycosylation Analysis

The major difference between glycoproteins produced in Chinese HamsterOvary (CHO) cell and plant cell systems is the glycosylation profile andglycan structure. Preliminary analysis has been performed tocharacterize the various N-linked glycan structures attached to theprotein. These results are compared to results of the N-glycosylationprofile found in commercial Enbrel. The presence of O-linked glycans,and glycan site analysis is determined.

Samples of prh TNFR2:FC and commercial Enbrel are reduced, alkylated andseparated on SDS-PAGE. The protein bands at ˜75 KDa (a total of about200 μg protein) are taken for glycan analysis using Trypsin digestionfollowed by either PNGase A or PNGase F digestion (˜80% and ˜20% of thetotal protein, respectively) for prh TNFR2:FC and PNGase F digestiononly for commercial Enbrel. Digestion with Trypsin, followed by PNGase Areleases all the N-linked glycans and digestion with PNGase F releasesall glycans except those containing alpha 1-3 core fucose (found inplants). The released glycans are extracted, cleaned and then labeledwith the fluorescent reagent anthranilamide (2-aminobenzamide, 2AB)followed by removal of excess 2AB. The analytical method includesseparation of the glycans on a Waters HPLC system with a normal phaseamide-based column (Tosoh TSK Amide-80 column), coupled with afluorescence detector (330 nm excitation, 420 nm emission). Sequencingof the labeled glycan pool is achieved by sequential digestion withvarious exoglycosidases followed by additional HPLC analysis. Usingsequential digestion with various exoglycosidases provides additionalinformation on the profile of the glycans structures and their relativeamounts. The exoglycosidase digestions that are carried out for theglycans released from prh TNFR2:FC are with JBH (Jack beanbeta-N-Acetylhexosaminidase) that removes beta 1-2, 3, 4 and 6N-acetylglucosamine (GlcNAc), with JBM (Jack bean mannosidase) thatremoves mannose alpha 1-2, 6>3 mannose and with BKF (Bovine testisfucosidase) that removes alpha 1-6 and alpha 1-3 core fucose. Thefluorescence labeling enables a semi-quantitative analysis of thedistribution of the various glycan structures in the total digestedglycan pool. The glycans are then separated according to unique glycanlinkages and in order of increasing size using a gradient solvent flowconsisting of ammonium formate and acetonitrile. Retention time ofindividual glycans is compared to the retention times of a standard mixof partially hydrolysed dextran fragments, giving a ladder of glucoseunits (GU). The glycans are assigned to peaks according to their GUvalues, based on standards and a comparison to an external data base(www(dot)glycobase(dot)nibrt(dot)ie:8080/database/show_glycobase(dot)action).The final assignment and relative peak areas are calculated from thechromatogram of the PNGase A digestion.

Enzyme-Linked Immunosorbent Assay (ELISA)

Binding ELISA:

TNFα binding ELISA is a combination of a commercial TNFα detection ELISAkit (Human TNF-α; Hycult Biotech Inc. #HK307) and a commercial antihuman IgG antibody (Goat anti human IgG FC specific HRP; Sigma). Theassay is a quantitative non radioactive assay for prhTNFR2:FC bindingactivity. This binding ELISA enables to detect functional (capable ofbinding TNFα) molecules comprising both the TNFR and IgG domains.

An ELISA plate pre-coated with antibodies against TNFα was incubatedwith TNFα (60 ng/ml, Sigma) for 1 hour at room temperature. Between eachELISA step the plate was washed three times with commercial wash buffer.Commercial Enbrel and supernatant from BY2 cells expressing prh TNFR2:FC(serial dilutions) were incubated on ELISA plate for 2 hr at RT. Goatanti human IgG Fc HRP was diluted 1:10,000 and incubated on plate for 1hr at RT. TMB was used as substrate for HRP. The colorimetric reactionwas stopped with 10% HCL and absorbance determined at 450 nm.

Prevention of TNF α Induced Apoptosis in A375 Cells

A375 cells (human melanoma cells) were grown in suspension in culturemedium (ATCC, #30-2002, supplemented with 10% FBS). 10⁴/well cells wereplated in 96-well assay plates and incubated overnight in assay medium(ATCC, #30-2002, supplemented with 5% FBS). Recombinant TNFα (2 ng/ml,ProSpec, Rehovot, Israel) was incubated for 2 hr at 37° C. in thepresence of different concentrations (1.562-100 ng/ml) of prhTNFR2:FC orcommercial Enbrel (Entanercept; Wyeth). Following incubation, the mixedsolution was added to A375 cells in the presence of actinomycin-D (0.8μg/ml), incubated for further 24 hr at 37° C., 5% CO₂ in a humidifiedincubator and quantification of apoptosis was determined by MTT assay(Sigma Cat. No. M5655). The plate was read at 570-650 nm and theinhibition of TNF-α induced cytotoxicity (%) was calculated.

Example 2 Protein Analysis

prhTNFR2:FC was analyzed under reducing (FIG. 2A) and non-reducingconditions (native extraction in the FIG. 2B). prhTNFR2:FC (Lane 1) andcommercial Enbrel (lane 2) were detected using anti Fc antibody (upperpanel) and anti TNFR2 antibody (lower panel). The two proteinsdemonstrate a slight difference in migration characteristics, presumablydue to differences in glycosylation patterns between the plant andmammalian cell-expressed enzymes.

TNFα binding by both commercial Enbrel and prh TNFR2:FC was examined bycomparing serial dilutions of lysates of BY2 cells expressing prhTNFR2:Fc (PRX-106) to commercial Enbrel. prh TNFR2:FC serial dilutionsdemonstrate a dose response binding pattern similar to the commercialprotein (see FIG. 3). The selection of transgenic cell lines accordingto protein expression was done by Western blotting. Thus, to allow forthe selection of individual cell lines, aliquots of highly diluted cellsuspension were spread on solid BY-2 medium. The cells were then grownuntil small calli developed. Each callus was then re-suspended in liquidculture. Cells were then sampled and evaluated for prh TNFR2:Fcexpression levels by extraction under reducing conditions followed byWestern Blot identification (anti FC antibody) of the produced targetprotein (FIG. 4). The functionality of the expressed protein wasestablished by its ability to prevent TNFα induced apoptosis.Specifically, TNFα activity can be measured by its ability to inducecell death of certain cell lines in the presence of the transcriptionalinhibitor, actinomycin D. Pre-incubation with a neutralizing protein ofTNFα prevents binding to the receptors (TNF-R1 and TNF-R2), therebyinhibiting the cytokine effect and preventing TNFα induced cell death.Quantification of cell viability by MTT assay provides an in-cellactivity assay for TNFα cytotoxicity. The results are shown in FIGS.5A-5G on melanoma cells A375 and in FIGS. 6A-6G on L929 fibroblasts.

Example 3 prh TNFR2:FC Suppresses Inflammatory Bowel Disease (IBD)

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatorycondition that is medicated by genetic, immune, and environmentalfactors. About 0.2-0.3% of the population are diagnosed with IBDannually. IBD is characterized by a tendency for chronic or relapsingimmune activation and inflammation within the Gastro-intestinal Tract(GIT). It has two presentations: Crohn's disease (CD), a chronicinflammation potentially involving any location of the GIT from mouth toanus, and Ulcerative colitis (UC), an inflammatory disorder that affectsthe rectum and extends proximally to affect variable extent of thecolon. CD is regulated more by the TH1 immune cell response,overproducing the cytokines IL-12, IFN-gamma and TNFalpha among others.UC, on the other hand, is mainly regulated by the TH2 immune cellresponse.

PRX106 is a soluble receptor for a cytokine overproduced in inflammationinvolving among others, the TH1 immune cell response. PRX106 was shownto be very effective when injected IV in other models of inflammation(Rheumatoid Arthritis). PRX106 is overexpressed in Protalix's ProCellEx™system, in BY2 plant cells. Being a plant cell, BY2 has a cell wall thatcan help protect PRX106 while moving through the stomach and smallintestine. In the colon, where the polysaccharides are digested, theplant cell releases its content and hence PRX106 is free to bind itscytokine ligand. Moreover, PRX106 is a chimeric protein carrying an Fcsegment of human IgG1. In the epithelial monolayer lining the mucosalbarrier, the FcRn receptor transcytoses IgG molecules across by bindingto their Fc. Therefore, PRX106 can also cross the epithelial barrier tobind its cytokine ligand on the serosal side of the epithelia.

IBD models are classified into five major groups: chemically inducedmodel, cell-transfer model, spontaneous model, congenital (spontaneousgene mutation) model, and genetically engineered model. In the mostwidely used chemically induced models, colitis is induced by intrarectaladministration of the covalently reactive reagents TNBS/oxazolone, whichare believed to induce a T-cell-mediated response againsthapten-modified autologous proteins/luminal antigens. In the DSS model,mice are subjected several days to drinking water supplemented with DSS(dextran sodium sulfate), which seems to be directly toxic to colonicepithelial cells of the basal crypts. The disease severity is evaluatedby scoring 3 major clinical signs (weight loss, diarrhea, and rectalbleeding). The mouse models TNBS (Example 3A) and DSS (Example 3B) areused to determine the therapeutic efficacy of the plant cells expressingthe chimeric polypeptide in vivo.

Example 3A PRX106-Expressing Cells are Effective in Alleviating Symptomsof IBD as Evidenced in an In Vivo Trinitrobenzene-Sulphonic Acid (TNBS)Model

Materials and Methods

Ethics Statement—

All procedures were strictly performed in accordance with the Guide forthe Care and Use of Laboratory Animals.

Animals

Male Balb/c mice, 8-9 weeks old were used in all experiments. Eachexperimental group included 5 to 10 mice. The mice were purchased fromHarlan Laboratories, Israel. All mice were moved to SPF-free room(natural bacterial flora) several days before starting the experiment.

TNBS Induction

TNBS was induced in mice by rectal installation of TNBS [M. F. Neurath,I. Fuss et al: j exp. Med 182′ 1281-1290 (1995)]. Mice were sensitizedby painting 100 μL of 1% TNBS in ethanol onto the shaved skin of theirabdomens 7 days before challenge. On the day of challenge, the mice weregiven 120 μL of 1% TNBS (Sigma Aldrich) slowly injected into the lumenof the colon via a catheter. Following TNBS treatment, mice were treatedper os (PO) daily from day 0 to day 4 with BY-2 cells expressingPRX-106, equivalent to 5 μg protein (Dose I) and 30 μg protein (DoseII); BY-2(−) control cells in the same orally administered volumes ofthe PRX-106 expressing cells; and saline. TNBS control mice received PBSalone. The animals were monitored once daily for weight. Weight loss wascalculated by subtracting the weight on each day from the weight on day0. After the experiment, the animals were sacrificed and dissected. Onday 5, blood samples were collected by cardiac puncture, and were leftto clot and then centrifuged to obtain serum for determination of serumcytokine levels. (Cury et al. Cell Immunol. 2013, 282 (1); 66-70.Experiments were performed on 5-15 mice per group in three separateexperiments; results followed the same pattern in all experiments.

Oral Administration of Recombinant Plant Cells

Oral administration of plant cells expressing recombinant TNFR2:Fc wasinitiated 6 hours after administration of TNBS. Mice received plantcells expressing recombinant TNFR2:Fc, resuspended in 350-500 μL.Negative controls received the same orally administered volumes of hostMock plant cells, instead of the plant cells expressing recombinantTNFR2:Fc. Oral administration was performed by gavage. Two more controlswere untreated mice and TNBS treated mice that received saline.

Analysis of Cytokine Profiles

Serum levels of cytokines TNF-α, and IL-10 were determined using ELISAkits following the manufacturer's instructions (R&D Systems,Minneapolis, Minn., USA).

Antibody Array

Serum qualitative measurement of cytokine content was performed usingthe Mouse Cytokine Antibody Array (R&D Systems, Minneapolis, Minn.,USA), according to the manufacturer's manual.

Immunohistochemistry

Paraffin-embedded colonic tissue sections (5 μm) were deparaffinized,rehydrated, washed and incubated in 3% H₂O₂ and blocked (Bar Sela et al2006). Slides were incubated with In-alpha pSer32/Ser36 antibodies(Abcam) Color was developed using the DAB substrate kit (ThermoScientific) or Zymed AEC substrate kit (Zymed Laboratories), followed bycounterstaining with Mayer's hematoxylin. Controls without addition ofprimary antibody showed low or no background staining in all cases.Blocking was performed according to Bar-Sela et al. Histopathology.2006; 49:188-193.

Flow Cytometry

Spleens were harvested from mice in RPMI 1640 medium. Cell suspensionswere prepared by dicing spleens with a razor blade, followed by passagethrough a 40 μM Nylon filter (BD Falcon). Splenocytes were incubatedwith anti-mouse CD4 (R&D Systems, Minneapolis, Minn., USA) andanti-mouse CD25 (R&D Systems, Minneapolis, Minn., USA). Cells were thenfixed and permeabilized for 20 min at 4° C. and then incubated withanti-mouse Foxp3 (Mouse Regulatory T cell 3-Color Flow kit, R&D Systems,Minneapolis, Minn., USA) diluted in permeabilization buffer for 30 min.Ten thousand CD4⁺ cells were analyzed by FACS.

Macroscopic Colon Damage

Macroscopic appearance of the colon was assessed using the Wallacemacroscopic scoring system [W. Vermeulen, J. G. de Man, S. Nullens, P.A. Pelckmans, B. Y. de Winter, and T. G. Moreels, “The use ofcolonoscopy to follow the inflammatory time course of TNBS colitis inrats,” Acta Gastro-Enterologica Belgica, vol. 74, no. 2, pp. 304-311,2011]. In this scoring system, the inflammation is assessed on thefollowing scale from 0 to 10 based on ulceration, inflammation, andextent of disease: 0=normal aspect of the mucosa, 1=localized hyperemiawithout ulcerations, 2=ulceration, 3=ulceration with thickening of bowelwall at one site, 4=two or more sites of ulceration and thickening ofthe bowel wall, 5=major sites of damage extending <2 cm along the lengthof the colon, and 6-10=damage extending >2 cm (with the score increasingby 1 for each centimeter of damaged tissue).

Results

Oral administration of plant cells expressing recombinant TNFR2:Fcimproved TNBS-Induced body weight loss as monitored 4 days afterinitiation of TNBS administration (FIGS. 7A-7B).

Colon lengths were measured as morphological indicators of coloninflammation in TNBS-treated mice; short colon indicating aninflammatory state. As shown in FIG. 8, the colon length of mice treatedwith TNBS was significantly shortened compared to the control mice. Thelength of colon of the treated group (oral administration of cellsexpressing prTNFR2:Fc) was significantly longer than that in theTNBS-treated group.

Oral administration of plant cells expressing recombinant TNFR2:Fc alsoimproved the macroscopic features of TNBS-induced colitis. Macroscopicexamination of colons, showed reduced colon damage severity comparedwith the non treated colons (FIG. 9).

Oral administration of plant cells expressing recombinant TNFR2:Fcreduced the expression of proinflammatory cytokines in mice withTNBS-induced colitis (FIGS. 10A-10C). The effect of the treatment on theserum levels of proinflammatory cytokines linked to TNBS colitis andanti-inflammatory cytokines was evaluated. Note that in most of theTNFR2:Fc treated groups the expression levels of proinflammatorycytokines IL-6 and TNF-α were reduced, and the expression levels ofanti-inflammatory cytokine IL-10 were elevated.

FIGS. 11A-11B showed that treatment with oral administration of plantcells expressing recombinant TNFR2:Fc reduced level of inflammatorymediators like granulocyte colony-stimulating factor G-CSF, macrophagecolony-stimulating factor (M-CSF), potentially indicating reducedsystemic inflammation by lowering systemic recruitment of bone marrowderived cells from the bloodstream.

Recently, imbalance of the development and function of IL-17-producingTh17 cells and CD4⁺CD25⁺FOXP3⁺ Treg cells has been demonstrated to playan important role in autoimmune diseases, including IBD. Treg cells,also known as CD4⁺CD25⁺, FOXP3⁺, are involved in the maintenance ofperipheral tolerance and in controlling the immune response byinitiating suppressive effects on activated immune cells. The presentanalysis shows that oral administration of plant cells expressingTNFR2:Fc expands population of functional regulatory T (T reg) cells inthe spleen (FIG. 12).

To conclude the above-results demonstrate that oral administration ofplant cells expressing recombinant TNFR2:Fc is an anti-inflammatoryagent that ameliorates TNBS-induced colitis.

Example 3B PRX106-Expressing Cells are Effective in Alleviating Symptomsof IBD as Evidenced in an In Vivo Dextran Sulfate Sodium-Induced(DSS-Induced) Model

The Dextran Sulfate Sodium-induced (DSS-induced) mouse model of IBD isused for compounds for efficacy in Inflammatory Bowel Disease. This isan experimental acute Ulcerative Colitis model with symptoms similar tothose observed in human UC, such as diarrhea, bloody feces, body weightloss, mucosal ulceration and shortening of the large intestine.

Ethics Statement

All procedures were strictly performed in accordance with the Guide forthe Care and Use of Laboratory Animals.

Animals

Male C67/Bl mice, 8-9 weeks old were used in all experiments. Eachexperimental group included 10 mice. The mice were purchased from HarlanLaboratories, Israel. All mice were moved to SPF-free room (naturalbacterial flora) several days before starting the experiment.

Induction and Evaluation of Colitis in Mice Treated with DSS andFollowing Oral Administration of Plant Cells Expressing RecombinantTNFR2:Fc

Colitis was induced by administration of 1.5% (wt/vol) DSS(reagent-grade DSS salt; molecular mass=36-50 kD; MP Biomedicals) innormal drinking water for 5 days, followed by 5 days of normal waterconsumption. Daily treatment with orally administered plant cellsexpressing recombinant TNFR2:Fc; Mock cells comprising vector alone orcontrol treatment (saline) began 24 hours following DSS induction, for aperiod of 7 days. Colonic inflammation was assessed 5 days after DSStreatment by punch biopsies and histological score. The animals weremonitored once daily for weight, weight loss was calculated bysubtracting the daily weight from the weight on day 0. After theexperiment, the animals were sacrificed and dissected; colon shorteningwas assessed by colon length measurements in comparison to untreatedcolon; Blood samples were collected by cardiac puncture, and were leftto clot and then centrifuged to obtain serum for determination of serumcytokine levels.

Oral Administration of Recombinant Plant Cells:

Oral administration of plant cells expressing recombinant TNFR2:Fc wasinitiated 24 hours following DSS administration. Mice received plantcells expressing recombinant TNFR2:Fc (comprising 30 μg of protein),suspended in 500 μl saline. Negative controls received the equivalentvolumes of host Mock plant cells, to the plant cells expressingrecombinant TNFR:Fc. Two more control groups were DSS treated miceadministered with saline and untreated mice. Oral administration wasperformed by gavage.

Analysis of Colon Inflammation.

Paraffin-embedded colon tissue sections were stained with hematoxylinand eosin for light microscopic examination to assess colon injury andinflammation. Samples from entire colon were analyzed pathologically bya pathologist blinded to treatment conditions. A scoring systemincluding degree of inflammation, crypt damage, percentage of areainvolved by inflammation and depth of inflammation was used.

Punch Biopsies.

Mouse colons were flushed 3 times with PBS containing antibiotics andopened along a longitudinal axis. Thereafter, 4-mm² punch biopsies wereobtained and incubated for 24 hours in RPMI-1640 medium supplementedwith antibiotics. Supernatants were collected and kept in −20° C. untilassessed for cytokine expression. Qualitative measurement of cytokinecontent in medium conditioned by colonic explants was performed usingthe Magnetic Luminex Screening Assay according to the manufacturer'smanual R&D Systems, Minneapolis, Minn., USA).

Analysis of Cytokine Profiles

Serum levels of cytokines TNF-α, IL-6 and IL-10 were determined usingMagnetic Luminex Screening Assay following the manufacturer'sinstructions (R&D Systems, Minneapolis, Minn., USA).

Results

1. Oral Administration of Plant Cells Expressing Recombinant TNFR2:FcImproved DSS-Induced Body Weight Loss

Body weight was monitored each day following DSS administration (FIGS.13A-13B). As can be seen, treatment of mice orally with plant cellsexpressing recombinant TNFR2:Fc attenuated the weight loss induced byDSS.

2. Oral Administration of Plant Cells Expressing Recombinant TNFR2:FcSuppressed DSS-Induced Colitis in Mice

Colon lengths were measured as it is well established that a short coloncan be used as a morphological indicator of colon inflammation inDSS-treated mice. As shown in FIGS. 14A-14B, the colon length of micetreated with DSS was significantly shortened compared with the controlmice. The length of colon in the oral administration of plant cellsexpressing recombinant TNFR2:Fc group was significantly longer than thatin the DSS-treated group.

3. Effect of Oral Administration of Plant Cells Expressing RecombinantTNFR2:Fc on Gut Inflammatory Cytokines Following DSS Colitis

FIGS. 15A-15C show a statistically significant decrease in gutproinflammatory cytokines following oral treatment with plant cellsexpressing recombinant TNFR2:Fc.

4. Oral Administration of Plant Cells Expressing Recombinant TNFR2:FcReduced the Expression of Proinflammatory Cytokines in Mice withDSS-Induced Colitis

The effect of Oral administration of plant cells expressing recombinantTNFR2:Fc on the production of proinflammatory cytokines linked to DSScolitis was evaluated. As shown in FIGS. 16A-16C, DSS induced proteinexpression of proinflammatory cytokines, such as IL-6 and TNF-α, insera, whereas Oral administration of plant cells expressing recombinantTNFR2:Fc suppressed the host protein secretion of proinflammatorycytokines. These results pointed out that oral administration of plantcells expressing recombinant TNFR2:Fc inhibited the production ofproinflammatory cytokine in the DSS-induced colitis model.

5. Histopathological Examination as an Indication for Colon Inflammation

The severity of colon inflammation was further evaluated by histologicalexaminations (FIGS. 17A-17B). Following DSS administration, colonsexhibited transmural inflammation and intense infiltration ofinflammatory cells. This cell influx associated with ulceration, loss ofgoblet cells and marked disruption in the crypts throughout the colon.On note, oral treatment with plant cells expressing TNFR2:Fc markedlyimproved the hisological features of DSS-induced colitis. Histologicalexamination of colons, showed reduced colon damage severity in colons oforally administered plant cells expressing recombinant TNFR2:Fc treatedmice, compared with the DSS and Mock treated colons.

In conclusion, the present study supports a role for orally administeredplant cells expressing recombinant TNFR2:Fc as an anti-inflammatoryagent with the capacity to ameliorate IBD.

Example 4 Evaluating the Recombinant TNFR2:Fc Protein PharmacokineticProfile in Rat Plasma at Various Time Points Post Feeding of Plant CellsExpressing Recombinant TNFR2:Fc

Materials and Methods

Animals

Rats (SD, females/9-10 weeks/n=6) were subject to a 20 hours fast andthen fed (free feeding) with cells expressing recombinant TNFR2:Fc(PRX-106) and host BY2(−). Following two hours from feeding, foodconsumption was measured. Young suckling rats (SD, males and females/16days/n=6) were 3 hours fasted and fed (by gavage) with cells expressingrecombinant TNFR2: Fc (PRX-106) and host BY2(−) cells.

Analysis of TNFR2 Profiles

Blood samples were collected in the each time point (e.g., 0, 1 h, 2 h,4 h, 6 h, 8 h, and 24 h), left to clot and then centrifuged to obtainserum. Serum levels of human TNFRII were determined using ELISA kitsfollowing the manufacturer's instructions (R&D Systems, Minneapolis,Minn., USA).

Results

TNFR2:Fc level in serum is shown in FIG. 18. Results demonstrate theelevation of TNFR2:Fc level in plasma following oral administration ofplant cells expressing the protein. TNFR2:Fc level in serum was detectedat 8 h, and was still detectable at 24 hours. TNFR2:Fc level in rat'sserum fed with host BY2(−) was not detectable.

The experiment was then followed by analysis of recombinant TNFR2:Fcprotein pharmacokinetic profile in the suckling rat plasma various timepoints post feeding of plant cells expressing TNFR2:Fc. TNFR2:Fc levelin serum is shown in FIG. 19. Results demonstrate the significantelevation of TNFR2:Fc level in plasma following oral administration ofTNFR2:Fc. TNFR2:Fc level in plasma peaked at 4 h. Probably, increasedlevel of TNFR2:Fc in serum of suckling rats relative to adult rat wasdue to expression of FcNR in intestine of suckling rats. TNFR2:Fc levelin rat's serum fed with host BY2(−) was not detectable.

Example 5 Toxicology Studies in Mice

Methods

Animals

Male and female SD Rats (Harlan Laboratories, Israel) 8 weeks at studyinitiation were housed under standard laboratory conditions. Mean weightat study initiation was approximately 6.8 gr for males and 6.3 gr forfemales. Animals were fed with commercial rodent diet (Teklad CertifiedGlobal 18% Protein Diet cat #: 2018SC) and had free access to autoclavedand acidified drinking water (pH between 2.5 and 3.5).

Study Design

Four groups, 3 dosing groups comprising 12 rats per group (6 males and 6females) and a control group comprising 6 rats per group (3 males and 3females), were assigned. In each gender, the control group receiveddilution buffer (0.2 M mannitol) and three treated groups received cellsexpressing TNFR2:Fc at dose levels of 0.1, 0.5 and 1 mg TNFR2:Fc/Kg bodyweight. Cells were aliquoted in accordance with requested expressedprotein amount. Each aliquot was mixed with 30 grams powder ofcommercial rodent diet and dilution buffer, to create a pellet. Thecontrol pellet was made with dilution buffer and commercial rodent dietpowder alone. All animals were daily orally fed with the pellets for 14days. During the study, mortality and general clinical observation wereperformed, bodyweight was monitored daily. At study termination (Day 15)after light anesthesia with carbon dioxide inhalation, three bloodsamples were drawn from all animals from the retro orbital sinus gross,after which, animals were sacrificed, pathology was executed andselected organs were harvested.

Results

No adverse clinical symptoms were recorded throughout the 14-day safetystudy. All blood parameters were within the normal range with nosignificant deviations. Body weight gain was persistent and normal withno significant difference between the groups (treated or Control). Cellsexpressing were found to be safe and well tolerated with no adverseeffects. No effect on biochemical parameters or clinical symptoms wasfound. Gross necropsy observation did not reveal pathological findings.No animal was found in a moribund state or under severe distressconditions. There were no observations of animals presenting severe painor decreased body weight.

Example 6 Sequencing of PRX-106

N Terminus Sequencing by Edman Degradation

Analysis was performed at Alphalyse (Denmark) uainf, an ABI Procise 494sequencer. The procedure determines the N-terminal amino acid sequenceof proteins and peptides by the Edman degradation chemistry. The Edmandegradation is a cyclic procedure where amino acid residues are cleavedoff one at a time and identified by chromatography. Here are 3 steps inthe cyclic procedure. In step 1, the PITC reagent is coupled to theN-terminal amino group under alkaline conditions. In step 2, theN-terminal residue is cleaved in acidic media. In step 3, the PITCcoupled residue is transferred to a flask, converted to a PTH-residueand identified by HPLC chromatography. The next cycle is then startedfor identification of the next N-terminal residue.

Results:

The sequence was determined to be LPAQV (SEQ ID NO: 18).

Amino Acid Sequence Verification by Reverse Phase HPLC Coupled to a MassSpectrometry Detector.

Sequencing was performed at the Smoler Proteomics Center(Technion—Israel Institute of Technology, Haifa, Israel). Analyses werecarried out using reverse-phase HPLC coupled to a mass spectrometrydetector.

Method

Proteolysis

The analyzed samples were resuspended in 8 M Urea, 100 mM ammoniumbicabonate (ABC) followed by reduction with 2.8 mM DTT (60° C. for 30min) and modified with 8.8 mM iodoacetamide in 100 mM ABC in the dark,at ambient temperature for an additional 30 min. The proteins weredigested overnight at 37° C. using modified trypsin (Promega) at a 1:50enzyme-to-substrate ratio in 2 M Urea, 25 mM ABC.

Mass Spectrometry Analysis

The tryptic or chymotryptic peptides were desalted using stage tips(home-made C18), the residual buffer was evaporated and the pellet wasresuspended in 0.1% (v/v) formic acid. Twenty nanogram of the resultingpeptides were resolved by reversed-phase liquid chromatography on a0.075×200-mm fused silica capillaries (J and W) packed with Reprosilreversed phase material (Dr Maisch GmbH, Germany). Peptides were elutedwith a linear 60 minutes gradient of 5 to 45% followed by 15 minutes at95% acetonitrile with 0.1% formic acid in water at flow rates of 0.25μL/min. On-line mass spectrometry was performed on an ion-trap massspectrometer (Orbitrap, Thermo) in a positive mode using repetitivelyfull MS scan followed by collision induced dissociation (CID) of the 7most dominant ions selected from the first MS scan. The massspectrometry data was analyzed using the Discoverer software version 1.3software using a specific protein derived database.

Results

The sequence was compared to the peptide sequence of the Etanerceptsequence. The identified sequences are presented in Table V, below.Presented is 84.8% coverage of the reference sequence (see green color,FIG. 20).

TABLE V Peptides Identified Following Digestion withTrypsin (SEQ ID NO: 19-203, ordered) WQQGnVFScSVMHEALHnHYTQKWQQGNVFScSVMHEALHNHYTqK GFYPSDIAVEWESNGqPENnYKT qYNSTYRVVSVLTVLHqDWLNGKWQqGNVFScSVMHEALHNHYTqKS VVSVLTVLHQDWLNGKEYKc VVSVLTVLHqDWLnGKEYKSqHTqPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPK WQQGnVFScSVMHEALHNHYScDKTHTcPPcPAPELLGGPSVFLFPPKPKD GQPREPqVYTLPPSREEMTKGFYPSDIAVEWESNGQPEnNYKT LPAqVAFTPYAPEPGSTcR EALHnHYTqKqNRIcTcRPGWYcALSKQEGcR WQQGNVFScSVmHEALHnHYTQKSqHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK GQPREPqVYTLPPSREEmTKGFYPSDIAVEWESnGQPENNYK SqHTQPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPKVVSVLTVLHQDWLnGK TYTqLWNWVPEcLScGSRcSSDqVETQAcTR WQQGNVFScSVMHEALHNHYTQKGFYPSDIAVEWESnGQPEnnYKT VVVDVSHEDPEVK PSTSFLLPMGPSPPAEGSTGDEPKLPAQVAFTPYAPEPGSTcR TTPPVLDSDGSFFL LSLSPGK EPQVYTLPPSREEMTKN SmAPGAVHLPQTTPPVLDSDGSFFLYSK WQQGNVFScSVmHEALHNHYTQK SMAPGAVH SVmHEALHNHYTQKVVSVLTVLH SQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPK GQPREPQVYAQVAFTPYAPEPGSTcR cAPLRK EPQVYTLPPSREEmTKnQVSLTcLVK SmAPGAVH VVSVLTVLHQDLFPPKPK GSFFLYSK IcTcRPGWY SQHTQPTPEPS SVLTVLHQDWLnGKEYK QVETQAcTRSLSLSPGK SDGSFFLYSK KALPAPIEK ALPAPIEK AVcTSTSPTR SQHTQPTPEPSTAPSTSFQVSLTcLVK LREYYDQTAqmccSKcSPGQHAK WQQGNVFScSVMHEALH DTLmISRPmGPSPPAEGSTGDEPK THTcPPcPAPELLGGPSVF DTLMISR SDQVETQAcTR KcRPGFGVARWYVDGVEVHNAK YVDGVEVHNAK TTPPVLDSDGSFF THTcPPcPAPELLGGPSVFLFPPKPKPSPPAEGSTGDEPK SLSLSPGKSEKD MAPGAVHLPQPVSTR VDGVEVHNAKScDKTHTcPPcPAPELLGGPSVF VVSVLTVLHQDWLNGK SLSLSPGKSEKPPcPAPELLGGPSVFLFPPKPK SFFLYSK FNWYVDGVEVHNAK FLLPMGPSPPAEGSTGDEPKDAVcTSTSPTR NQVSLTcLVK NqVSLTcLVKG SLSPGKSEK TPEVTcVVVDVSHEDPEVKLREYYDQTAQM GFYPSDIAVEWESNGQPENNYK FNWYVDGVEVHN VVSVLTVLHQDWLNSQHTQPTPEPSTAPST RTPEVTcVVVDVSHEDPEVK SLSLSPGKS LSPGKSEKDEL LPQPVSTRTTPPVLDSDGSFFLY TSDTVcDScEDSTYTQLWN ALPAQVAFTPYAPEPGSTcR EEQYNSTYRScDKTHTcPPcPAPELLGGPSVFLFPPKPK cSPGQHAKVFcTK TPEVTcVVVDVSHEDSMAPGAVHLPQPV TcRPGWYcALSK TcPPcPAPELLGGPSVFLFPPKPKTSDTVcDScEDSTYTQLWNWVPEcLScGSR LcAPLRK SPPAEGSTGDEPK WVPEcLScGSRGPSPPAEGSTGDEPK SSDQVETQAcTR EEQYnSTYR VAFTPYAPEPGSTcR PGWYcALSKcRPGFGVAR ScSVmHEALHnHYTqK VVSVLTVLHQDWLNGKEYK LcAPLREPQVYTLPPSREEMTKnQVSLTcLVK LLPMGPSPPAEGSTGDEPKSQHTQPTPEPSTAPSTSFLLPmGPSPPAEGSTGDEPK SLSLSPGKSE EEMTKNqV SVMHEALHNHYTQKSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKScDK EEmTKnQVSLTcLVKGLREYYDQTAQmccSK cSSDqVETQAcTR EPQVYTLPPSREEMTK NQVSLTcLVKG cSSDQVETQAcTRnQVSLTcLVK TKPREEQYNSTYR PAQVAFTPYAPEPGSTcR SLSLSPGKSEKDELAFTPYAPEPGSTcR APGAVHLPQPVSTR SDGSFFLYSKLTVDK THTcPPcPAPELLGVVSVLTVLHQDWLn EPQVYTLPPSR SmAPGAVHLPQPVSTR GQPREPQVYTLPPSREEmTKTPYAPEPGSTcR EVTcVVVDVSHEDPEVK TKPREEQYnSTYR VSnKALPAPIEKLREYYDQTAQMccSK FTPYAPEPGSTcR SMAPGAVHLPQPVSTR GPSVFLFPPKPKVVSVLTVLHQDWLnGKEYK SQHTQPTPEPSTAPS SMAPGAVHLPQPVS AVHLPQPVSTRGQPREPQVYTLPPSR PGAVHLPQPVSTR TLMISR KNqVSLTcLVKGFYPSDIAVEWESNGqPENnYKLREYYDQTAQMc SmAPGAVHLPQPV LPAPIEK EYYDQTAQMccSK NWVPEcLScGSRSLSPGKSEKDEL IcTcRPGWYcALSK SMAPGAVHLPQPVST EYYDQTAQmccSK ASMDAVcTSTSPTRSQHTQPTPEPSTAPSTS TLPPSREEMTK SQHTQPTPEPSTAPSTSFL TLmISREPQVYTLPPSREEmTK GQPREPQVYTLPPSREEMTK TPEVTcVVVDVSHEDPEVKFNScDKTHTcPPcPAPELLG GFYPSDIAVEWESNGqPENnYK AKGQPREPQVYTLPPSRLREYYDQTAQMcc LPmGPSPPAEGSTGDEPK ScSVMHEALHNHYTQK FNWYVDGVEVHnAKPMGPSPPAEGSTGDEPK SMAPGAVHLPqPVSTR SMAPGAVHLPQ LPMGPSPPAEGSTGDEPK

Example 7 Evaluation of Orally Administered PRX-106 Plant CellsExpressing Recombinant TNFR2:Fc in the Clinical Context

In order to assess safety of administration of orally administered plantcells expressing recombinant TNFR2:Fc, also referred to herein asOPRX-106 and to evaluate pharmacokinetic parameters of the OPRX-106,OPRX-106 is administered to healthy subjects as further described hereinbelow.

Primary Objective

The primary objective of this trial is to evaluate the safety ofOPRX-106, in healthy male volunteers.

Secondary Objective

The secondary objective of this clinical study is to evaluate thepharmacokinetics of TNFR2-Fc following administration of OPRX-106 inhealthy male volunteers.

Overall Study Design and Plan—Description

This is a Phase 1, randomized, open-label study to evaluate the safetyof OPRX-106 and pharmacokinetics of TNFR-Fc following oraladministration of OPRX-106 in healthy volunteers. Up to 18 healthy malesubjects (age 18 years and older) are randomly allocated to one of threedose cohorts (up to 6 and not less than 4 subjects per cohort) receivingOPRX-106 doses equivalent to 2 mg, 8 mg or 16 mg TNFR-Fc. Subjectsreceive daily oral administrations of OPRX-106 for 5 consecutive days.Subjects remain at the clinical center for 12 hours after the firstadministration of OPRX-106 and then return to the site as needed foradditional administrations and study procedures. The cells are providedas a cell suspension.

At screening and on Days 1 (0 and 24 hours), 5 and 10, vital signs aremeasured, a physical examination is effected and blood samples areretrieved for laboratory tests. Adverse events (AEs) and concomitantmedications are evaluated at all study visits.

Blood samples for Plasma TNFR-Fc levels are drawn three times (in 2 hourintervals) at screening, on Days 1 and 4: pre-administration, 2, 4, 6,8, 10, 12, ±15 minutes at each time point, and 24±2 hours postadministration.

A control (placebo) group is not necessary, as all safety endpoints areassessed as change from baseline. Subjects are allocated to a dosecohort to limit bias but the study is open-label in order to identifyany dose-dependent adverse effects of OPRX-106.

Selection of Study Population

Inclusion Criteria

To be eligible for the study, subjects must meet the inclusion criterialisted below:

1. Healthy male age 18 or older;2. BMI 20-25 kg/m²;3. Male subjects or their partners must use an adequate method ofcontraception at all times during the study;4. Negative laboratory tests for HIV, HBcAb and anti HCV at thescreening visit;5. Naïve to any previous recombinant protein therapy;6. Provide written informed consent; and7. Have the ability to understand the requirements of the study and tocomply with the study protocol and dosing regimen.

Exclusion Criteria

To be eligible for the study, subjects must not meet any of theexclusion criteria listed below:

1. Clinical evidence of any active significant disease that couldpotentially compromise the ability of the Investigator to evaluate orinterpret the effects of the study treatment on safety assessment, thusincreasing the risk to the subject to unacceptable levels;2. Presence of any acute or chronic diseases;3. History of any allergies or protein-drug hypersensitivity;4. Suspected Tuberculosis (previous, active or latent);5. Exposure to long-term steroid treatment within the last 12 monthsprior to the study;6. Subject had a major operation in last 6 months;7. Subject has received immunosuppressive treatment;8. Chronic use of any medication including vitamins;9. Participation in another clinical trial during the previous 3 months(subject report);10. Reported history of alcohol or drug abuse;11. Subjects with short bowel (more than 1 m removed of small bowel);

Removal of Subjects from Therapy or Assessment

Reasons for permanent discontinuation include the following:

Clinically significant adverse events (AEs);

Serious protocol violation;

Sponsor elects to terminate the study;

Subject requires the use of prohibited treatment or medication;

Intolerance to treatment;

The subject experiences progressive or severe hypersensitivity—to betreated appropriately and withdrawn from the study;

The subject requests to discontinue treatment or withdraws consent;

The Investigator feels that it is not in the best interest of thesubject to continue treatment and/or if the Investigator believes thatthe subject can no longer be compliant with the requirements of thestudy;

Replacement Policy

Withdrawn subjects may be replaced upon Medical Director decision.

Primary Endpoint

The safety of each of the three doses of OPRX-106 as assessed by AEs,physical examinations, vital signs, concomitant medications andlaboratory test results.

Vital signs are measured at screening and on Days 1 (0 pre-dose and 24hours), 5 and 10. Vital signs comprising systolic and diastolic arterialblood pressure, pulse and body temperature are assessed at all studyvisits. Systolic and diastolic blood pressure is measured after thesubject has been in supine position for at least 1 minute. Allrecordings are performed using standard equipment. Clinicallysignificant abnormal findings are reported as AEs.

Clinical Laboratory Tests—Blood samples for clinical laboratory safetytests are collected after 8 hours fasting, at screening and pre-doseadministration on Days 1, 2, and 5, and on Day 10. Clinical laboratorytests include total protein, albumin, alanine transaminase (ALT),aspartate aminotransferase (AST), alkaline phosphatase (ALP),gamma-glutamyl transpeptidase (GGTP), Lactate dehydrogenase (LDH),cholesterol, uric acid, creatinine, blood urea nitrogen (BUN), sodium(Na), potassium (K), fasting glucose, total bilirubin, and completeblood cell count (CBC) with differential. Clinically significantabnormal findings are reported as AEs.

Secondary Endpoint

The PK profile of TNFR-Fc following administration of each of the threedoses of OPRX-106 on the first (Day 1) and last (Day 4) ofadministration as assessed by AUC_(0-t), C_(max), t_(max) and additionalPK parameters based on observed results.

PK Parameters

Blood samples for Plasma TNFR-Fc levels are drawn three times (in 2 hourintervals) at screening, on Day 1 and 4: pre-administration, 2, 4, 6, 8,10, 12, ±15 minutes at each time point, and 24±2 hours postadministration.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. An oral dosage form comprising the plant cellscomprising a nucleic acid construct having a nucleic acid sequenceencoding a plant produced chimeric polypeptide comprising: (i) a firstdomain which comprises a TNFα binding domain of a TNF receptor, and (ii)a second domain which comprises an Fc domain of an immunoglobulin,wherein said first domain and said second domain are N-terminally toC-terminally respectively sequentially translationally fused and whereinthe chimeric polypeptide specifically binds TNFα, the oral dosage formcomprising 1-20 mg of said polypeptide.
 2. The oral dosage form of claim1 comprising 1-10 mg of said polypeptide.
 3. The oral dosage form ofclaim 1 comprising 10-20 mg of said polypeptide.
 4. The oral dosage formof claim 1, wherein said chimeric polypeptide comprises an additionaldomain encoding an endoplasmic reticulum signal peptide translationallyfused N-terminally to said first domain.
 5. The oral dosage form ofclaim 4, wherein said signal peptide is a plant signal peptide.
 6. Theoral dosage form of claim 5, wherein said plant signal peptide is as setforth in SEQ ID NO:
 4. 7. The oral dosage form of claim 1, wherein saidfirst domain is 200-250 amino acids long.
 8. The oral dosage form ofclaim 7, wherein said first domain comprises the amino acid sequenceLCAP (SEQ ID NO: 11) and VFCT (SEQ ID NO: 12).
 9. The oral dosage formof claim 8, wherein said first domain further comprises the amino acidsequence LPAQVAFXPYAPEPGSTC (SEQ ID NO: 13).
 10. The oral dosage form ofclaim 9, wherein said first domain is as set forth in SEQ ID NO:
 2. 11.The oral dosage form of claim 1, wherein said immunoglobulin is IgG₁.12. The oral dosage form of claim 1, wherein said second domain is asset forth in SEQ ID NO:
 9. 13. The oral dosage form of claim 1, whereinsaid chimeric polypeptide is as set forth in SEQ ID NO:
 6. 14. The oraldosage form of claim 1, wherein said chimeric polypeptide is as setforth in SEQ ID NO:
 7. 15. The oral dosage form of claim 1, wherein saidchimeric polypeptide comprises a plant-specific glycan.
 16. A method oftreating a TNFα-associated medical condition in a subject in needthereof, the method comprising orally administering to the subject atherapeutically effective amount of the oral dosage form of claim
 1. 17.The method of claim 16, wherein said medical condition is aninflammatory disease.
 18. The method of claim 16, wherein said medicalcondition is an autoimmune disease.
 19. The method of claim 16, whereinsaid medical condition is selected from the group consisting ofrheumatoid arthritis, ankylosing spondyloarthritis, plaque psoriasis,rheumatoid polyarthritis, psoriatic arthritis, ankylosingspondyloarthritis and juvenile idiopathic arthritis.
 20. The method ofclaim 16, wherein said medical condition is an inflammatory boweldisease.